Inducing remission of Type 2 diabetes in the Caribbean: findings from a mixed methods feasibility study of a low-calorie liquid diet-based intervention in Barbados.

AIM: In a high proportion of people with recently diagnosed Type 2 diabetes, a short (2-3-month) low-calorie diet is able to restore normal glucose and insulin metabolism. The aim of this study was to determine the feasibility of this approach in Barbados. METHODS: Twenty-five individuals with Type 2 diabetes diagnosed within past 6 years, not on insulin, BMI ≥ 27 kg/m2 were recruited. Hypoglycaemic medication was stopped on commencement of the 8-week liquid (760 calorie) diet. Insulin response was assessed in meal tests at baseline, 8 weeks and 8 months. Semi-structured interviews, analysed thematically, explored participants' experiences. 'Responders' were those with fasting plasma glucose (FPG) < 7 mmol/l at 8 weeks. RESULTS: Ten men and 15 women (mean age 48, range 26-68 years) participated. Mean (sd) BMI was 34.2 kg/m2 (6.0); FPG 9.2 mmol/l (2.2). Mean weight loss at 8 weeks and 8 months was 10.1 kg [95% confidence interval (CI) 8.1, 12.0] and 8.2 kg (95% CI 5.8, 10.6); FPG was lower by 2.2 mmol/l (95% CI 1.2, 3.2) and 1.7 mmol/l (95% CI 0.8, 2.7) respectively. Nine of 11 (82%) of those who lost ≥ 10 kg were 'responders' compared with 6 of 14 (43%) who lost < 10 kg (P = 0.048). The 30-min insulin increment was higher in responders at baseline and follow-up (P ≤ 0.01). A food culture based on starchy foods and pressures to eat large amounts at social events were among the challenges identified by participants. CONCLUSIONS: The feasibility of this approach to weight loss and diabetes remission in a predominantly black population in Barbados was demonstrated.

The prevalence of peripheral neuropathy severe enough to cause a loss of protective sensation in a population-based sample of people with known and newly detected diabetes in Barbados: a cross-sectional study.

AIMS: To determine the prevalence and potential risk factors for diabetic peripheral neuropathy with a loss of protective sensation in Barbados. METHODS: A representative population sample aged > 25 years with previously diagnosed diabetes or a fasting blood glucose ≥ 7 mmol/l or HbA1c ≥ 48 mmol/mol (6.5%) was tested by 10 g monofilament at four plantar sites per foot and a 28 Hz tuning fork and neurothesiometer at the hallux. Data were adjusted to the age structure of people with diabetes in Barbados. Multivariable logistic regression assessed associations with peripheral neuropathy with a loss of protective sensation. RESULTS: Of 236 participants [74% response rate, 33% men, 91% black, median age 58.6 years, mean BMI 30.1 kg/m2 , mean HbA1c 54 mmol/mol (7.1%)], 51% had previously diagnosed diabetes. Foot examination demonstrated that 25.8% (95% CI 20.2 to 31.5) had at least one insensate site with monofilament testing, 14.8% (95% CI 10.2 to 19.4) had an abnormal tuning fork test and 10.9% (95% CI 6.9 to 14.9) had a vibration perception threshold > 25 V. Peripheral neuropathy with a loss of protective sensation prevalence was 28.5% (95% CI 22.7 to 34.4) as indicated by monofilament with ≥ 1 insensate site and/or vibration perception threshold > 25 V. With previously diagnosed diabetes the prevalence was 36.4% (95% CI 27.7 to 45.2) with 98.4% of cases identified by monofilament testing. Increasing age, previously diagnosed diabetes, male sex and abdominal obesity were independently associated with peripheral neuropathy with a loss of protective sensation. CONCLUSIONS: Over a third of people with previously diagnosed diabetes had evidence of peripheral neuropathy with a loss of protective sensation. Monofilament testing alone may be adequate to rule out peripheral neuropathy with a loss of protective sensation. Monofilament and neurothesiometer stimuli are reproducible but dependent on participant response.

Deriving an optimal threshold of waist circumference for detecting cardiometabolic risk in sub-Saharan Africa.

BACKGROUND: Waist circumference (WC) thresholds derived from western populations continue to be used in sub-Saharan Africa (SSA) despite increasing evidence of ethnic variation in the association between adiposity and cardiometabolic disease and availability of data from African populations. We aimed to derive a SSA-specific optimal WC cut-point for identifying individuals at increased cardiometabolic risk. METHODS: We used individual level cross-sectional data on 24 181 participants aged ⩾15 years from 17 studies conducted between 1990 and 2014 in eight countries in SSA. Receiver operating characteristic curves were used to derive optimal WC cut-points for detecting the presence of at least two components of metabolic syndrome (MS), excluding WC. RESULTS: The optimal WC cut-point was 81.2 cm (95% CI 78.5-83.8 cm) and 81.0 cm (95% CI 79.2-82.8 cm) for men and women, respectively, with comparable accuracy in men and women. Sensitivity was higher in women (64%, 95% CI 63-65) than in men (53%, 95% CI 51-55), and increased with the prevalence of obesity. Having WC above the derived cut-point was associated with a twofold probability of having at least two components of MS (age-adjusted odds ratio 2.6, 95% CI 2.4-2.9, for men and 2.2, 95% CI 2.0-2.3, for women). CONCLUSION: The optimal WC cut-point for identifying men at increased cardiometabolic risk is lower (⩾81.2 cm) than current guidelines (⩾94.0 cm) recommend, and similar to that in women in SSA. Prospective studies are needed to confirm these cut-points based on cardiometabolic outcomes.International Journal of Obesity advance online publication, 31 October 2017; doi:10.1038/ijo.2017.240.

A checklist that enhances the provision of education during insulin initiation simulation: a randomized controlled trial.

AIM: The study tested the hypothesis that doctors using an insulin information checklist during simulated insulin initiation would impart more information regarding insulin use. METHODS: A total of 128 simulations were conducted. Doctors (n = 64) were recruited from practitioners recently completing internship (n = 19) and those established in primary care (n = 45). Both groups of doctors were strata randomized to control (n = 32) and intervention groups (n = 32), so that each group contained equal numbers. Doctors in each group experienced two identical simulations of insulin initiation with an intervening period of 10 min. Doctors in the intervention arm were introduced to an insulin initiation checklist, which they reviewed independently and utilized in the second simulation. Trained assessors captured the provision of education in 21 predefined educational areas. Differences in the change of the total education provided between the first and second simulations were assessed using linear regression. RESULTS: The difference in the mean change of education provided between the first and second simulations within the 21 educational areas for the control and treatment groups was 9.7 [95% confidence interval (CI): 8.8-11.1, P < 0.001] - an increase of 46.2%. The difference for the 15 areas relevant to pen use was 7.3 (95% CI: 6.2-8.4, P < 0.001) - an increase of 51.6%. CONCLUSIONS: The checklist resulted in doctors providing significantly more education applicable to syringe and insulin pen routes of insulin administration during simulations. Further research is needed on the checklist's impact on healthcare professionals and patient outcomes in the clinical context. (Clinical Trials Registry No: NCT02266303).

Systems Science for Caribbean Health: the development and piloting of a model for guiding policy on diabetes in the Caribbean.

BACKGROUND: Diabetes is highly prevalent in the Caribbean, associated with a high morbidity and mortality and is a recognised threat to economic and social development. Heads of Government in the Caribbean Community came together in 2007 and declared their commitment to reducing the burden of non-communicable diseases (NCDs), including diabetes, by calling for a multi-sectoral, systemic response. To facilitate the development of effective policies, policymakers are being engaged in the development and use of a system dynamics (SD) model of diabetes for Caribbean countries. METHODS: Previous work on a diabetes SD model from the United States of America (USA) is being adapted to a local context for three countries in the region using input from stakeholders, a review of existing qualitative and quantitative data, and collection of new qualitative data. Three country models will be developed using one-on-one stakeholder engagement and iterative revision. An inter-country model will also be developed following a model-building workshop. Models will be compared to each other and to the USA model. The inter-country model will be used to simulate policies identified as priorities by stakeholders and to develop targets for prevention and control. The model and model-building process will be evaluated by stakeholders and a manual developed for use in other high-burden developing regions. DISCUSSION: SD has been applied with success for health policy development in high-income country settings. The utility of SD in developing countries as an aid to policy decision-making related to NCDs has not been tested. This study represents the first of its kind.

Undiagnosed diabetes in the over-60s: performance of the Association of Public Health Observatories (APHO) Diabetes Prevalence Model in a general practice.

AIM: The Association of Public Health Observatories (APHO) Diabetes Prevalence Model has been interpreted to suggest that a substantial number of people with diabetes are 'missed'. An affluent suburb of Newcastle upon Tyne has a low known diabetes prevalence. We aimed to determine the true prevalence of diabetes in the practice population aged over 60 years and compare our prevalence estimate with that of the Association of Public Health Observatories Diabetes Prevalence Model (18.0%; uncertainty limit 10.7-27.7%). At baseline, the known prevalence of diabetes in this group was 7.4%. METHODS: All individuals aged 60 years and over registered with one general practice in Newcastle-upon-Tyne, not known to have diabetes (n = 1375), were invited for a standard oral glucose tolerance test and measurement of HbA(1c). Standard World Health Organization cut points for fasting and post-challenge glucose on oral glucose tolerance test or HbA(1c) ≥ 48 mmol/mol (6.5%) were used to identify diabetes. RESULTS: Five hundred and eighty-four individuals (42.5%) attended for screening. Using oral glucose tolerance test criteria, 4.5% were identified with undiagnosed diabetes. Using HbA(1c), 3.1% had undiagnosed diabetes. The estimated prevalence of total diabetes for the practice population aged 60 years and older is 11.8 (10.5-13.2%) and 10.3 (9.3-11.6) for oral glucose tolerance test and HbA(1c) criteria, respectively. CONCLUSIONS: The prevalence of diabetes in those aged 60 years and older registered with this practice is lower than the point estimate of the Association of Public Health Observatories Diabetes Prevalence Model, but within its uncertainty limits. Application of the Association of Public Health Observatories model must take into account its uncertainty limits.

Dutch versus English advantage in the epidemic of central and generalised obesity is not shared by ethnic minority groups: comparative secondary analysis of cross-sectional data.

BACKGROUND: Ethnic minority groups in Western European countries tend to have higher levels of overweight than the majority populations for reasons that are poorly understood. Investigating relative differences between countries could enable an investigation of the importance of national context in determining these inequalities. OBJECTIVE: To explore: (1) whether Indian and African origin populations in England and the Netherlands are similarly disadvantaged compared with the White populations in terms of the prevalence of overweight and central obesity; (2) whether the previously known Dutch advantage of relatively low overweight prevalence is also observed in Dutch ethnic minority groups and (3) the contribution of health behaviour and socio-economic position to the differences observed. METHODS: Secondary analyses of population-based studies of 16 406 participants from England and the Netherlands. Prevalence ratios were estimated using regression models. RESULTS: Except for African men, ethnic minority groups in both countries had higher rates of overweight and central obesity than their White counterparts. However, the Dutch minority groups were relatively more disadvantaged than English minority groups as compared with the majority populations. The Dutch advantage of the low prevalence of obesity was only seen in White men and women and African men. In contrast, English-Indian (prevalence ratio=0.87, 95% confidence interval (CI): 0.81-0.93) and English-Caribbean (prevalence ratio=0.82, 95% CI: 0.76-0.89) women were less centrally obese than their Dutch equivalents. The Dutch-Indian men were very similar to the English-Indian men. The contribution of health behaviour and socio-economic position to the observed differences were small. CONCLUSION: Contrary to the patterns in White groups, the Dutch ethnic minority women were more obese than their English equivalents. More work is needed to identify factors that may contribute to these observed differences.

Chronic non-communicable disease risk factor survey 2010 among University of the West Indies staff at Cave Hill, Barbados.

OBJECTIVE: To determine the prevalence of risk factors for chronic non-communicable diseases (CNCDs) among staff of The University of the West Indies (UWI), Cave Hill campus, in Barbados. METHODS: A self-administered questionnaire comprising validated questions from the WHO STEPS NCD Risk Factor Survey, the Jamaica Healthy Lifestyle (JHL) Survey and the Behaviour Risk Factor (BRF) Survey, was conducted during the Staff Health Day in May 2010, and at four locations on campus during July 2010. Standardized measurements of weight, height and blood pressure were taken. Data were analysed using EXCEL and STATA and results were compared to the Barbados 2007 STEPS NCD survey. RESULTS: The target population was all staff at the Cave Hill campus of UWI. The coverage rate was 25.2% (269/1068); 63.8% of males and 75% of females were either overweight or obese. Ninety-seven per cent ate less than the recommended 5 fruits and vegetables per day. Low levels of physical activity were reported in 51.9% of males and 62.2% of females. Thirty-two per cent of males and 13% of females were binge drinkers. All participants had at least one of the risk factors (current daily smoker < 5 fruits and vegetables/day, physical inactivity, overweight/obese and raised blood pressure) whilst 48% of males and 57.2% of females demonstrated three or more risk factors. These results are similar to those found in the Barbados STEPS NCD risk factor survey of 2007. CONCLUSION: The results confirm a similar high prevalence of NCD risk factors among Cave Hill UWI staff as among the Barbadian population. The study reveals opportunities to inform policy on strategies to positively impact the risk factors.

Three-dimensional structure of the acetylcholine receptor by cryoelectron microscopy and helical image reconstruction.

Long tubular vesicles have been grown from isolated Torpedo postsynaptic membranes, in which the receptors are arranged helically on the vesicle surface. The structures of these tubes have been analyzed by cryoelectron microscopy of specimens embedded in thin films of ice, combined with helical image reconstruction. Complete data sets from tubes belonging to several helical families have been obtained to a resolution of 17 A in all directions. Confirming a preliminary study (Toyoshima, C., and N. Unwin. 1988. Nature (Lond.). 336:247-250), the central ion channel has an almost constant diameter throughout the molecule except for the portion extending through the hydrophobic part of the lipid bilayer, where the pore is too small to be resolved. However, the density on the pseudo fivefold axis running through the pore is consistently highest in the cytoplasmic half of the bilayer, suggesting the gate is located in that region. The path followed by each subunit has been identified throughout the length of the receptor. The two alpha subunits follow equivalent paths. All subunits have similar features which change in character at the same level relative to the membrane.

Location of subunits within the acetylcholine receptor by electron image analysis of tubular crystals from Torpedo marmorata.

The binding sites on the nicotinic acetylcholine receptor of labels specific for the alpha-, beta-, and delta-subunits were determined by electron image analysis, using tubular crystals of receptors grown from the postsynaptic membranes of Torpedo marmorata electric organ. The labels were alpha-bungarotoxin (which attaches to the acetylcholine binding sites on the pair of alpha-subunits), Fab35 (a monoclonal antibody Fab fragment directed against the main immunogenic region of the alpha-subunit), Fab111 (a monoclonal antibody Fab fragment directed against a cytoplasmic site on the beta-subunit), and wheat germ agglutinin (which binds to N-acetylglucosamine residues on the delta-subunit). These labels, bound to receptors in the crystals, were located by comparing labeled with native structures, averaged in each case over more than 5,000 molecules. From the assignments made, we find that the clockwise arrangement of subunits around the receptor, viewed from the synaptic face, is: alpha, beta, alpha, gamma, and delta; that the main immunogenic region is at (or close to) the side of the alpha-subunit; and that the two acetylcholine binding sites are at the synaptic end of the alpha-subunits, 27-28 A from the central axis and approximately 53 A apart. In the crystal lattice, neighboring molecules are paired so that their delta- and alpha-subunits are juxtaposed, an organization that appears to relate closely to the grouping of receptors in vivo.

Arrangement of the acetylcholine receptor subunits in the resting and desensitized states, determined by cryoelectron microscopy of crystallized Torpedo postsynaptic membranes.

Two conformational states of the nicotinic acetylcholine receptor have been investigated by cryoelectron microscopy of flattened vesicular crystals grown from Torpedo marmorata postsynaptic membranes. One was obtained from the vesicles without acetylcholine present, and is presumed to correspond to the native, or resting state; the other was obtained from the vesicles after exposure to 100 microM to 5 mM carbamylcholine (an acetylcholine analogue) and is presumed to correspond to a desensitized state. Both conformations were determined in three-dimensions to a resolution of 18 A, sufficient to reveal the configurations of the five subunits around the central ion channel over most of their length. The subunits of either structure have a similar appearance, consistent with their amino acid homology. They are each aligned almost parallel to the axis of the receptor, conferring a high degree of pentagonal symmetry to the bilayer portion and a contiguous region on the synaptic side. Their external surfaces form a pronounced ridge in the bilayer portion, which broadens toward the synaptic end. Comparison of features in the two three-dimensional maps reveals that carbamylcholine induces a quaternary rearrangement, involving predominantly the delta-subunit. The densities corresponding to this subunit are tilted by approximately 10 degrees tangential to the axis of the receptor over a large fraction of its length, and become misaligned relative to the densities corresponding to the other four subunits. The gamma-subunit is also affected, being displaced slightly away from the axis of the receptor. The alpha- and beta-subunits may be affected on a more localized scale. The overall changes are most pronounced in the synaptic region, where the ligand-binding site is located, and in the cytoplasmic region, which may be closer to the gate of the channel. The physiological process of desensitization appears to be associated with a structural transition in which the subunits switch to a less symmetrical configuration.

Isolation and purification of gap junction channels.

This paper reports methods we have developed to solubilize gap junction channels, or connexons, from isolated gap junctions and to purify them in milligram quantities. Two sources of material are used: rat liver gap junctions and gap junctions produced by infecting insect cells with a baculovirus containing the cDNA for human liver beta 1 protein (connexin 32). Complete solubilization is obtained with long chain detergents (lauryl dimethyl amineoxide, dodecyl maltoside) and requires high ionic strength and high pH as well as reducing conditions. The purification involves chromatography on hydroxylapatite and gel filtration on Superose 6. A homogeneous product is indicated by a single band on a silver-stained gel and a homogeneous population of doughnut-shaped particles under the electron microscope. These particles have hexameric symmetry. The purified connexons have a tendency to form aggregates: filaments and sheets. The filaments grow by end-to-end association of connexons and are nonpolar, suggesting that the connexons are paired as in the cell-to-cell channel. The sheets grow by lateral association of the filaments.

Adverse risk factor trends limit gains in coronary heart disease mortality in Barbados: 1990-2012.

BACKGROUND: Although most countries face increasing population levels of obesity and diabetes their effect on coronary heart disease (CHD) mortality has not been often studied in small island developing states (SIDs) where obesity rates are among the highest in the world. We estimated the relative contributions of treatments and cardiovascular risk factors to the decline in CHD mortality from 1990 to 2012 in the Caribbean island, Barbados. METHODS: We used the IMPACT CHD mortality model to estimate the effect of increased coverage of effective medical/surgical treatments and changes in major CHD risk factors on mortality trends in 2012 compared with 1990. We calculated deaths prevented or postponed (DPPs) for each model risk factor and treatment group. We obtained data from WHO Mortality database, population denominators from the Barbados Statistical Service stratified by 10-year age group (ages 25-34 up to 85 plus), population-based risk factor surveys, Global Burden of Disease and Barbados' national myocardial infarction registry. Monte Carlo probabilistic sensitivity analysis was performed. RESULTS: In 1990 the age-standardized CHD mortality rate was 109.5 per 100,000 falling to 55.3 in 2012. Implementation of effective treatment accounted for 56% DPPs (95% (Uncertainty Interval (UI) 46%, 68%), mostly due to the introduction of treatments immediately after acute myocardial infarction (AMI) (14%) and unstable angina (14%). Overall, risk factors contributed 19% DPPs (95% UI 6% to 34%) mostly attributed to decline in cholesterol (18% DPPs, 95% UI 12%, 26%). Adverse trends in diabetes: 14% additional deaths(ADs) 95% UI 8% to 21% ADs) and BMI (2% ADs 95%UI 0 to 5% ADs) limited potential for risk factor gains. CONCLUSIONS: Given the significant negative impact of obesity/diabetes on mortality in this analysis, research that explores factors affecting implementation of evidenced-based preventive strategies is needed. The fact that most of the decline in CHD mortality in Barbados was due to treatment provides an example for SIDs about the advantages of universal access to care and treatment.

Three-dimensional location of the main immunogenic region of the acetylcholine receptor.

About two-thirds of the antibodies to the nicotinic acetylcholine (ACh) receptor in patients with the autoimmune disease myasthenia gravis bind to the main immunogenic region (MIR). This is small, well-defined region on each of the two alpha subunits, containing residues 67-76 (alpha 67-76). By determining the structure of the ACh receptor complexed with two different fragments of an MIR-directed antibody, we have determined the three-dimensional location of the MIR (and therefore residues alpha 67-76) to be at the extreme synaptic end of each alpha subunit. The antibody fragments extend from the binding site away from the receptor axis and into the synaptic cleft, minimizing any steric interference neighboring ACh receptors might have with their binding.

Images of purified Shaker potassium channels.

BACKGROUND: Voltage-gated K+ channels play an important role in the control of neuronal excitability and synaptic plasticity. Their low abundance and extraordinary heterogeneity have rendered their purification from natural sources difficult. We have previously cloned a voltage-gated K(+)-channel gene, Shaker, from Drosophila. The Shaker K(+)-channel polypeptide resembles one of the four internal repeats of a Na(+)- or Ca(2+)-channel alpha subunit, suggesting that this example of a K+ channel contains four identical or homologous subunits. Similar K(+)-channel polypeptides have been characterized from mammals, other vertebrate and invertebrate species, and from plants. Electrophysiological studies of K+ channels expressed in Xenopus oocytes suggest that they are indeed tetramers, and heteromultimeric K+ channels have been found in the mammalian brain. Until now, however, no K+ channel, nor any other member of the superfamily of voltage-gated ion channels, has been characterized by electron microscopy or other structural analysis. RESULTS: We have purified Shaker K+ channels, expressed in insect Sf9 cells, to apparent homogeneity, and imaged them using the electron microscope. The physical dimensions of these molecules, as well as their biochemical characteristics, are consistent with a tetrameric subunit composition. Moreover, the Shaker channel revealed by negative staining has the appearance of a four-fold symmetric tetramer, with a large, central vestibule that presumably constitutes part of the pathway for ions. CONCLUSION: These first clear images of a voltage-gated ion channel reveal a marked four-fold symmetry. The integrity of the purified tetrameric complex indicates that the purification scheme used in this study may be further developed for future structural analysis of voltage-gated K+ channels.

Macromolecular structure determination by electron microscopy: new advances and recent results.

Electron microscopy is undergoing a mini-renaissance, as a number of biological systems are yielding to higher resolution analysis as a result of advances in instrumentation, specimen preparation and image-processing technology. The atomic structure of tubulin has now been solved, crucial elements of secondary structure have recently been revealed in several membrane proteins (rhodopsin, gap junctions, aquaporin, and Ca2+ and H+ ATPases) and in a virus particle, and macromolecular complexes are being seen in increasingly fine detail. This growth has been enhanced further by the ability to combine structures of macromolecular complexes derived by electron microscopy with X-ray structures of their components, in order to reconstruct molecular machines and large multiprotein complexes in immense detail.

Role of GTP hydrolysis in microtubule dynamics: information from a slowly hydrolyzable analogue, GMPCPP.

The role of GTP hydrolysis in microtubule dynamics has been reinvestigated using an analogue of GTP, guanylyl-(alpha, beta)-methylene-diphosphonate (GMPCPP). This analogue binds to the tubulin exchangeable nucleotide binding site (E-site) with an affinity four to eightfold lower than GTP and promotes the polymerization of normal microtubules. The polymerization rate of microtubules with GMPCPP-tubulin is very similar to that of GTP-tubulin. However, in contrast to microtubules polymerized with GTP, GMPCPP-microtubules do not depolymerize rapidly after isothermal dilution. The depolymerization rate of GMPCPP-microtubules is 0.1 s-1 compared with 500 s-1 for GDP-microtubules. GMPCPP also completely suppresses dynamic instability. Contrary to previous work, we find that the beta--gamma bond of GMPCPP is hydrolyzed extremely slowly after incorporation into the microtubule lattice, with a rate constant of 4 x 10(-7) s-1. Because GMPCPP hydrolysis is negligible over the course of a polymerization experiment, it can be used to test the role of hydrolysis in microtubule dynamics. Our results provide strong new evidence for the idea that GTP hydrolysis by tubulin is not required for normal polymerization but is essential for depolymerization and thus for dynamic instability. Because GMPCPP strongly promotes spontaneous nucleation of microtubules, we propose that GTP hydrolysis by tubulin also plays the important biological role of inhibiting spontaneous microtubule nucleation.

Nicotinic acetylcholine receptor at 9 A resolution.

The nicotinic acetylcholine receptor is a cation-selective, ligand-gated ion channel, involved in signal transmission at the chemical synapse. This paper reports the three-dimensional appearance of the channel in the closed conformation, at 9 A resolution. The structure was determined by electron microscopy of tubular crystals of Torpedo postsynaptic membranes embedded in amorphous ice. The analysis was carried out by averaging data from separate images, using helical diffraction methods. The images were recorded over a wide range of defocus (7000 to 18,800 A) so that all spacings in the object were well sampled. Tubes of only one kind ((-16.6) helical family) were processed, so that the Fourier terms could be averaged directly in reciprocal space. The three-dimensional map, obtained from 26 images, resolves some elements of secondary structure within the five protein subunits. In the synaptic part of each subunit, about 30 A from the bilayer surface, there is a group of three rods that are oriented predominantly perpendicular to the plane of the bilayer and twist around each other as in a left-handed coil. These rods presumably are alpha-helices. Two of them line the entrance to the channel, and the third is on the outside. The distinctive appearance of the alpha subunits in this region suggests that the rods may be involved in forming the binding pocket for acetylcholine. In the bilayer-spanning part of each subunit there is only one rod clearly visible, which forms the wall lining the pore, and so is assumed to be the transmembrane helix, M2. This rod does not form a straight path through the lipid bilayer, but bends, or kinks, near its mid-point, where it is closest to the axis of the pore, and tilts radially outwards on either side. It is flanked on the lipid-facing sides by a continuous rim of density, which seems likely to be composed of beta-sheet. A tentative alignment is made between the three-dimensional densities and the sequence of M2, based on correlation of the appearance of the rods with a special pattern of amino acid residues in the sequence. This alignment places the charged groups at the ends of M2 symmetrically on either side of the bilayer, and a highly conserved leucine residue (Leu251 of the alpha subunit) at the level of the kink.(ABSTRACT TRUNCATED AT 400 WORDS)

Projection structure of the nicotinic acetylcholine receptor: distinct conformations of the alpha subunits.

The nicotinic acetylcholine (ACh) receptor is a neurotransmitter-gated ion channel consisting of a ring of five membrane-spanning subunits, including two (the alpha subunits) with identical amino acid sequences. To open the channel ACh has to bind at two sites, involving both alpha subunits, which have widely different affinities. An earlier three-dimensional electron microscopic study of the non-activated Torpedo receptor had suggested that these sites might be cavities with the alpha subunits, located 25 to 30 A from the membrane, and hence that the different affinities might be associated with alternative conformations of the alpha subunits. This paper compares the conformations of the alpha subunits by determining the projection structure of the non-activated receptor and correlating the projection and three-dimensional maps. The projection structure was calculated to 7.5 A resolution from images of ice-embedded "giant" tubular crystals of Torpedo membranes, which had been partially flattened to make pairs of two-dimensional sheets (rho 2 lattice: a=90.2 A, beta=162.3 A, gamma=121.7 degrees). Each subunit in projection occupied a sector of approximately 72 degrees, and had a peak of high density at a distance of 22 to 25 A from the pseudo 5-fold axis. This peak in the single subunit between the alphas was approximately 3 A further from the axis than were the other peaks, and the projected density distributions composing the two alpha subunits were different. Close matching of the projected densities with the three-dimensional densities 25 to 35 A from the membrane, showed unequivocally that corresponding (alpha-helical) rods, which encircle the cavities in the two alpha subunits, are unequally inclined. Therefore, the cavities are indeed shaped by distinct conformations of the alpha subunits before ACh has bound. A model is proposed of how both alpha subunits participate concertedly to open the channel, assuming that ACh binds to these internal sites.

Electron microscopic evidence for the assembly of soluble pentameric extracellular domains of the nicotinic acetylcholine receptor.

Exploitation of soluble extracellular domains (ECDs) of the nicotinic acetylcholine receptor may provide a route to crystallographic studies aimed at exploring the structure and function of the intact receptor. The first step towards this goal is to manufacture and isolate soluble fragments that fold and assemble to form a functionally relevant complex. The baculovirus insect cell expression system was used to co-express soluble ECDs of all four muscle-type nicotinic acetylcholine receptor subunits (alpha, beta, gamma & delta-ECD) from Torpedo. Protein complexes were purified using either the conformationally sensitive monoclonal antibody mAb35, specific for a folded alpha subunit, or a NiNTA affinity resin, specific for a polyhistidine tag engineered on the delta-ECD. Western blotting with subunit specific antibodies confirmed the co-expression of each ECD and furthermore, indicated that the alpha, beta and gamma-ECDs were being co-purified with the polyhistidine-tagged delta-ECD. Chemical cross-linking was used to show that these co-purified proteins had indeed interacted specifically to form soluble oligomeric complexes. A low-resolution, three-dimensional image of these purified complexes, composed only of ECDs, was obtained by electron microscopy. They were shown to resemble the extracellular vestibule of the native receptor, having the same pseudo-pentameric symmetry, size and shape. Expression of incomplete sets of the four nicotinic acetylcholine receptor ECDs did not yield detectable complexes.