Structure of the C-terminal domain of the Prokaryotic Sodium Channel Orthologue NsvBa

Crystallographic and electrophysiological studies have recently provided insight into the structure, function and drug binding of prokaryotic sodium channels. These channels exhibit significant sequence identities, especially in their transmembrane regions, with human voltage-gated sodium channels. However, rather than being single polypeptides with four homologous domains, they are tetramers of single domain polypeptides, with a C-terminal domain (CTD) composed of an inter-subunit four helix coiled-coil. The structures of the CTDs differ between orthologues. In NavBh and NavMs, the C-termini form a disordered region adjacent to the final transmembrane helix, followed by a coiled-coil region, as demonstrated by synchrotron radiation circular dichroism (SRCD) and double electron-electron resonance electron paramagnetic resonance spectroscopic measurements. In contrast, in the crystal structure of the NavAe orthologue, the entire C-terminus is comprised of a helical region followed by a coiled-coil. In this study we have examined the CTD of the NsvBa from Bacillus alcalophilus, which unlike other orthologues is predicted by different methods to have different types of structures: either a disordered adjacent to the transmembrane region, followed by a helical coiled-coil, or a fully helical CTD. To discriminate between the two possible structures we have used SRCD spectroscopy to experimentally determine the secondary structure of the C-terminus of this orthologue and used the results as the basis for modelling the transition between open and closed conformations of the channel

Differential lipid dependence of the function of bacterial sodium channels

The lipid bilayer is important for maintaining the integrity of cellular compartments and plays a vital role in providing the hydrophobic and charged interactions necessary for membrane protein structure, conformational flexibility and function. To directly assess the lipid dependence of activity for voltage-gated sodium channels, we compared the activity of three bacterial sodium channel homologues (NaChBac, NavMs, and NavSp) by cumulative 22Na+ uptake into proteoliposomes containing a 3:1 ratio of 1-palmitoyl 2-oleoyl phosphatidylethanolamine and different “guest” glycerophospholipids. We observed a unique lipid profile for each channel tested. NavMs and NavSp showed strong preference for different negatively-charged lipids (phosphatidylinositol and phosphatidylglycerol, respectively), whilst NaChBac exhibited a more modest variation with lipid type. To investigate the molecular bases of these differences we used synchrotron radiation circular dichroism spectroscopy to compare structures in liposomes of different composition, and molecular modeling and electrostatics calculations to rationalize the functional differences seen. We then examined pore-only constructs (with voltage sensor subdomains removed) and found that in these channels the lipid specificity was drastically reduced, suggesting that the specific lipid influences on voltage-gated sodium channels arise primarily from their abilities to interact with the voltage-sensing subdomains

Characterization of the Prokaryotic Sodium Channel NavSp Pore with a Microfluidic Bilayer Platform

This paper describes the use of a newly-developed micro-chip bilayer platform to examine the electrophysiological properties of the prokaryotic voltage-gated sodium channel pore (NavSp) from Silicibacter pomeroyi. The platform allows up to 6 bilayers to be analysed simultaneously. Proteoliposomes were incorporated into suspended lipid bilayers formed within the microfluidic bilayer chips. The chips provide access to bilayers from either side, enabling the fast and controlled titration of compounds. Dose-dependent modulation of the opening probability by the channel blocking drug nifedipine was measured and its IC50 determined

Structure of a bacterial voltage-gated sodium channel pore reveals mechanisms of opening and closing

Voltage-gated sodium channels are vital membrane proteins essential for electrical signalling; in humans, they are key targets for the development of pharmaceutical drugs. Here we report the crystal structure of an open-channel conformation of NavMs, the bacterial channel pore from the marine bacterium Magnetococcus sp. (strain MC-1). It differs from the recently published crystal structure of a closed form of a related bacterial sodium channel (NavAb) by having its internal cavity accessible to the cytoplasmic surface as a result of a bend/rotation about a central residue in the carboxy-terminal transmembrane segment. This produces an open activation gate of sufficient diameter to allow hydrated sodium ions to pass through. Comparison of the open and closed structures provides new insight into the features of the functional states present in the activation cycles of sodium channels and the mechanism of channel opening and closing

Characteristics of Nondisabled Older Patients Developing New Disability Associated with Medical Illnesses and Hospitalization

OBJECTIVE: To identify demographic, clinical, and biological characteristics of older nondisabled patients who develop new disability in basic activities of daily living (BADL) during medical illnesses requiring hospitalization. DESIGN: Longitudinal observational study. SETTING: Geriatric and Internal Medicine acute care units. PARTICIPANTS: Data are from 1,686 patients aged 65 and older who independent in BADL 2 weeks before hospital admission, enrolled in the 1998 survey of the Italian Group of Pharmacoepidemiology in the Elderly Study. MEASUREMENTS: Study outcome was new BADL disability at time of hospital discharge. Sociodemographic, functional status, and clinical characteristics were collected at hospital admission; acute and chronic conditions were classified according to the International Classification of Disease, ninth revision; fasting blood samples were obtained and processed with standard methods. RESULTS: At the time of hospital discharge 113 patients (6.7%) presented new BADL disability. Functional decline was strongly related to patients’ age and preadmission instrumental activities of daily living status. In a multivariate analysis, older age, nursing home residency, low body mass index, elevated erythrocyte sedimentation rate, acute stroke, high level of comorbidity expressed as Cumulative Illness Rating Scale score, polypharmacotherapy, cognitive decline, and history of fall in the previous year were independent and significant predictors of BADL disability. CONCLUSION: Several factors might contribute to loss of physical independence in hospitalized older persons. Preexisting conditions associated with the frailty syndrome, including physical and cognitive function, comorbidity, body composition, and inflammatory markers, characterize patients at high risk of functional decline

Mutagenesis of the NaChBac sodium channel discloses a functional role for a conserved S6 asparagine

Asparagine is conserved in the S6 transmembrane segments of all voltage-gated sodium, calcium, and TRP channels identified to date. A broad spectrum of channelopathies including cardiac arrhythmias, epilepsy, muscle diseases, and pain disorders is associated with its mutation. To investigate its effects on sodium channel functional properties, we mutated the simple prokaryotic sodium channel NaChBac. Electrophysiological characterization of the N225D mutant reveals that this conservative substitution shifts the voltage-dependence of inactivation by 25 mV to more hyperpolarized potentials. The mutant also displays greater thermostability, as determined by synchrotron radiation circular dichroism spectroscopy studies of purified channels. Based on our analyses of high-resolution structures of NaChBac homologues, we suggest that the side-chain amine group of asparagine 225 forms one or more hydrogen bonds with different channel elements and that these interactions are important for normal channel function. The N225D mutation eliminates these hydrogen bonds and the structural consequences involve an enhanced channel inactivation

Regulation of Fission Yeast Morphogenesis by PP2A Activator pta2

Cell polarization is key for the function of most eukaryotic cells, and regulates cell shape, migration and tissue architecture. Fission yeast, Schizosaccharomyces pombe cells are cylindrical and polarize cell growth to one or both cell tips dependent on the cell cycle stage. Whereas microtubule cytoskeleton contributes to the positioning of the growth sites by delivering polarity factors to the cell ends, the Cdc42 GTPase polarizes secretion via actin-dependent delivery and tethering of secretory vesicles to plasma membrane. How growth is restricted to cell tips and how re-initiation of tip growth is regulated in the cell cycle remains poorly understood. In this work we investigated the function of protein phosphatase type 2A (PP2A) in S. pombe morphogenesis by deleting the evolutionary conserved PTPA-type regulatory subunit that we named pta2. pta2-deleted cells showed morphological defects and altered growth pattern. Consistent with this, actin patches and active Cdc42 were mislocalized in the pta2 deletion. These defects were additive to the lack of Cdc42-GAP Rga4. pta2Δ cells show upregulated Cdc42 activity and pta2 interacts genetically with polarisome components Tea1, Tea4 and For3 leading to complete loss of cell polarity and rounded morphology. Thus, regulation of polarity by PP2A requires the polarisome and involves Pta2-dependent control of Cdc42 activity

Positive Darwinian Selection in the Piston That Powers Proton Pumps in Complex I of the Mitochondria of Pacific Salmon

The mechanism of oxidative phosphorylation is well understood, but evolution of the proteins involved is not. We combined phylogenetic, genomic, and structural biology analyses to examine the evolution of twelve mitochondrial encoded proteins of closely related, yet phenotypically diverse, Pacific salmon. Two separate analyses identified the same seven positively selected sites in ND5. A strong signal was also detected at three sites of ND2. An energetic coupling analysis revealed several structures in the ND5 protein that may have co-evolved with the selected sites. These data implicate Complex I, specifically the piston arm of ND5 where it connects the proton pumps, as important in the evolution of Pacific salmon. Lastly, the lineage to Chinook experienced rapid evolution at the piston arm

Quantitative proteomics of delirium cerebrospinal fluid

Delirium is a common cause and complication of hospitalization in older people, being associated with higher risk of future dementia and progression of existing dementia. However relatively little data are available on which biochemical pathways are dysregulated in the brain during delirium episodes, whether there are protein expression changes common among delirium subjects and whether there are any changes which correlate with the severity of delirium. We now present the first proteomic analysis of delirium cerebrospinal fluid (CSF), and one of few studies exploring protein expression changes in delirium. More than 270 proteins were identified in two delirium cohorts, 16 of which were dysregulated in at least 8 of 17 delirium subjects compared with a mild Alzheimer's disease neurological control group, and 31 proteins were significantly correlated with cognitive scores (mini-mental state exam and acute physiology and chronic health evaluation III). Bioinformatics analyses revealed expression changes in several protein family groups, including apolipoproteins, secretogranins/chromogranins, clotting/fibrinolysis factors, serine protease inhibitors and acute-phase response elements. These data not only provide confirmatory evidence that the inflammatory response is a component of delirium, but also reveal dysregulation of protein expression in a number of novel and unexpected clusters of proteins, in particular the granins. Another surprising outcome of this work is the level of similarity of CSF protein profiles in delirium patients, given the diversity of causes of this syndrome. These data provide additional elements for consideration in the pathophysiology of delirium as well as potential biomarker candidates for delirium diagnosis