Diabetes mellitus and intermittent claudication: a cross-sectional study of 920 claudicants

INTRODUCTION: Diabetes mellitus (DM) and intermittent claudication (IC) are frequently associated health conditions. Our hypothesis is that the nature, severity and quality of life (QoL) of patients with IC and DM are worse than those of claudicant patients without diabetes. MATERIAL AND METHODS: An observational, cross-sectional and multicentre study of 920 patients with IC, divided into two groups: diabetic (n = 477) and non-diabetic (n = 443). For each group, we examined clinical and biological characteristics (including levels of glucose and lipids), the ankle-brachial index (ABI), responses to the Walking Impairment Questionnaire (WIQ) and the European Quality of Life-5 Dimensions (EQ-5D) questionnaire. RESULTS: Compared with claudicant patients without diabetes, claudicants with diabetes were older (p < 0.001), more likely to be female (p = 0.006), with a higher BMI (p < 0.001), more likely to have a sedentary lifestyle (p < 0.001) and to be a non-smoker (p < 0.001). Claudicant patients with diabetes also had significantly more cardiovascular risk factors (p < 0.001), more frequent ischaemic cardiopathy (p = 0.023) and chronic renal failure (p = 0.002), and fewer prior ictus events (p = 0.003). No significant differences between groups were found with respect to blood pressure, levels of cholesterol or triglycerides. The mean ABI of diabetic-IC patients was slightly lower than IC patients without diabetes (p = 0.016). All WIQ subdomains scores were significantly lower (p < 0.001), indicating poorer walking ability, in claudicant and diabetic patients with compared with those without diabetes. The mean E5-QD global scores and the mean EQ-5D visual analogue scale in the whole series were 0.58 (SD = 0.21) and 55.04 (SD = 21.30), respectively. Both E5-QD scores were significantly lower, indicating poorer QoL, in claudicant patients with diabetes than claudicant patients without diabetes (p < 0.001). CONCLUSION: Patients with IC and DM had more risk cardiovascular factors, cardiovascular conditions, disability and worse haemodynamic status and QoL than claudicant patients without diabetes

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.This work was partly supported by grants PGC2018-093505-B-I00 from the Spanish “Ministerio de Ciencia e Innovación”/FEDER, UE, and FCT-Fundação para a Ciência e a Tecnologia, I.P., under the scope of the project UIDB/04565/2020 and UIDP/04565/2020 of the Research Unit Institute for Bioengineering and Biosciences—iBB and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB

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.The work in the authors’ laboratories has been supported by grants SAF2017-82977-P (AEI/FEDER, UE) and PGC2018-093505-B-I00 from MINECO and GRE17-01 from Universidad de Alicante (Spain)

Muscle-Type Nicotinic Receptor Blockade by Diethylamine, the Hydrophilic Moiety of Lidocaine

Lidocaine bears in its structure both an aromatic ring and a terminal amine, which can be protonated at physiological pH, linked by an amide group. Since lidocaine causes multiple inhibitory actions on nicotinic acetylcholine receptors (nAChRs), this work was aimed to determine the inhibitory effects of diethylamine (DEA), a small molecule resembling the hydrophilic moiety of lidocaine, on Torpedo marmorata nAChRs microtransplanted to Xenopus oocytes. Similarly to lidocaine, DEA reversibly blocked acetylcholine-elicited currents (IACh) in a dose-dependent manner (IC50 close to 70 μM), but unlike lidocaine, DEA did not affect IACh desensitization. IACh inhibition by DEA was more pronounced at negative potentials, suggesting an open-channel blockade of nAChRs, although roughly 30% inhibition persisted at positive potentials, indicating additional binding sites outside the pore. DEA block of nAChRs in the resting state (closed channel) was confirmed by the enhanced IACh inhibition when pre-applying DEA before its co-application with ACh, as compared with solely DEA and ACh co-application. Virtual docking assays provide a plausible explanation to the experimental observations in terms of the involvement of different sets of drug binding sites. So, at the nAChR transmembrane (TM) domain, DEA and lidocaine shared binding sites within the channel pore, giving support to their open-channel blockade; besides, lidocaine, but not DEA, interacted with residues at cavities among the M1, M2, M3, and M4 segments of each subunit and also at intersubunit crevices. At the extracellular (EC) domain, DEA and lidocaine binding sites were broadly distributed, which aids to explain the closed channel blockade observed. Interestingly, some DEA clusters were located at the α-γ interphase of the EC domain, in a cavity near the orthosteric binding site pocket; by contrast, lidocaine contacted with all α-subunit loops conforming the ACh binding site, both in α-γ and α-δ and interphases, likely because of its larger size. Together, these results indicate that DEA mimics some, but not all, inhibitory actions of lidocaine on nAChRs and that even this small polar molecule acts by different mechanisms on this receptor. The presented results contribute to a better understanding of the structural determinants of nAChR modulation.This work was supported by grants BFU2012-31359, BFU2012-39092-C02-01, BFU2011-25920, and CSD2008-00005 from the MINECO and PROMETEO/2014/11 from GVA (Spain)

Structural stabilization of botulinum neurotoxins by tyrosine phosphorylation

AbstractTyrosine phosphorylation of botulinum neurotoxins augments their proteolytic activity and thermal stability, suggesting a substantial modification of the global protein conformation. We used Fourier-transform infrared (FTIR) spectroscopy to study changes of secondary structure and thermostability of tyrosine phosphorylated botulinum neurotoxins A (BoNT A) and E (BoNT E). Changes in the conformationally-sensitive amide I band upon phosphorylation indicated an increase of the α-helical content with a concomitant decrease of less ordered structures such as turns and random coils, and without changes in β-sheet content. These changes in secondary structure were accompanied by an increase in the residual amide II absorbance band remaining upon H-D exchange, consistent with a tighter packing of the phosphorylated proteins. FTIR and differential scanning calorimetry (DSC) analyses of the denaturation process show that phosphorylated neurotoxins denature at temperatures higher than those required by non-phosphorylated species. These findings indicate that tyrosine phosphorylation induced a transition to higher order and that the more compact structure presumably imparts to the phosphorylated neurotoxins the higher catalytic activity and thermostability

Filling Single-Walled Carbon Nanotubes with Lutetium Chloride : A Sustainable Production of Nanocapsules Free of Nonencapsulated Material

Filled carbon nanotubes are of interest for a wide variety of applications ranging from sensors to magnetoelectronic devices and going through the development of smart contrast and therapeutic agents in the biomedical field. In general, regardless of the method employed, bulk filling of carbon nanotubes results in the presence of a large amount of external nonencapsulated material. Therefore, further processing is needed to achieve a sample in which the selected payload is present only in the inner cavities of the nanotubes. Here, we report on a straightforward approach that allows the removal of nonencapsulated compounds in a time efficient and environmentally friendly manner, using water as a "green" solvent, while minimizing the residual waste. The results presented herein pave the way toward the production of large amounts of high-quality closed-ended filled nanotubes, also referred to as carbon nanocapsules, readily utilizable in the foreseen applications

N-type inactivation of the potassium channel KcsA by the Shaker B "ball" Peptide: Mapping the inactivating peptide-binding epitope

10 pags, 6 figsThe effects of the inactivating peptide from the eukaryotic Shaker B K + channel (the ShB peptide) on the prokaryotic KcsA channel have been studied using patch clamp methods. The data show that the peptide induces rapid, N-type inactivation in KcsA through a process that includes functional uncoupling of channel gating. We have also employed saturation transfer difference (STD) NMR methods to map the molecular interactions between the inactivating peptide and its channel target. The results indicate that binding of the ShB peptide to KcsA involves the ortho and meta protons of Tyr 8, which exhibit the strongest STD effects; the C4H in the imidazole ring of His16; the methyl protons of Val4, Leu 7, and Leu10 and the side chain amine protons of one, if not both, the Lys18 and Lys19 residues. When a noninactivating ShB-L7E mutant is used in the studies, binding to KcsA is still observed but involves different amino acids. Thus, the strongest STD effects are now seen on the methyl protons of Val4 and Leu10, whereas His16 seems similarly affected as before. Conversely, STD effects on Tyr8 are strongly diminished, and those on Lys18 and/or Lys19 are abolished. Additionally, Fourier transform infrared spectroscopy of KcsA in presence of 13C-labeled peptide derivatives suggests that the ShB peptide, but not the ShB-L7E mutant, adopts a β-hairpin structure when bound to the KcsA channel. Indeed, docking such a β-hairpin structure into an open pore model for K+ channels to simulate the inactivating peptide/channel complex predicts interactions well in agreement with the experimental observations. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.This work was supported by Spanish Ministerio de Educación y Ciencia Grants CTQ2005-00360/BQU (to J. L. N.) and BFU2005-00749 (to J. M. G.-R.);FIPSE Experiment 36557/06 (to J. L. N.) and Grant BANCAJA-UMH IP/UR/01;and Consellería de Empresa, Universidad y Ciencia de la Generalitat Valenciana Grant GV07/017 (to J. A. E.)

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 work was supported by grants BFU2012-31359, BFU2015-66612-P, SAF2015-66275-C2-1-R and SAF2017-82977-P (AEI/FEDER, UE) from MINECO, PROMETEO/2014/11 from Generalitat Valenciana (Spain) and GRE17-01 from Universidad de Alicante. R.C. held a predoctoral fellowship from Universidad de Alicante (FPUUA36) and M.N. a predoctoral industrial fellowship from Ministerio de Economía, Industria y Competitividad (DI-16-08303)

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.This work was partly supported by the grant BFU2015-66612-P from the Spanish MINECO/FEDER, UE. CD-G acknowledges support from Medical Biochemistry and Biophysics Doctoral Programme (M2B-PhD) and FCT Portugal (SFRH/PD/BD/135154/2017)

Muscle-Type Nicotinic Receptor Modulation by 2,6-Dimethylaniline, a Molecule Resembling the Hydrophobic Moiety of Lidocaine

To identify the molecular determinants responsible for lidocaine blockade of muscle-type nAChRs, we have studied the effects on this receptor of 2,6-dimethylaniline (DMA), which resembles lidocaine’s hydrophobic moiety. Torpedo marmorata nAChRs were microtransplanted to Xenopus oocytes and currents elicited by ACh (IACh), either alone or co-applied with DMA, were recorded. DMA reversibly blocked IACh and, similarly to lidocaine, exerted a closed-channel blockade, as evidenced by the enhancement of IACh blockade when DMA was pre-applied before its co-application with ACh, and hastened IACh decay. However, there were marked differences among its mechanisms of nAChR inhibition and those mediated by either the entire lidocaine molecule or diethylamine (DEA), a small amine resembling lidocaine’s hydrophilic moiety. Thereby, the IC50 for DMA, estimated from the dose-inhibition curve, was in the millimolar range, which is one order of magnitude higher than that for either DEA or lidocaine. Besides, nAChR blockade by DMA was voltage-independent in contrast to the increase of IACh inhibition at negative potentials caused by the more polar lidocaine or DEA molecules. Accordingly, virtual docking assays of DMA on nAChRs showed that this molecule binds predominantly at intersubunit crevices of the transmembrane-spanning domain, but also at the extracellular domain. Furthermore, DMA interacted with residues inside the channel pore, although only in the open-channel conformation. Interestingly, co-application of ACh with DEA and DMA, at their IC50s, had additive inhibitory effects on IACh and the extent of blockade was similar to that predicted by the allotopic model of interaction, suggesting that DEA and DMA bind to nAChRs at different loci. These results indicate that DMA mainly mimics the low potency and non-competitive actions of lidocaine on nAChRs, as opposed to the high potency and voltage-dependent block by lidocaine, which is emulated by the hydrophilic DEA. Furthermore, it is pointed out that the hydrophobic (DMA) and hydrophilic (DEA) moieties of the lidocaine molecule act differently on nAChRs and that their separate actions taken together account for most of the inhibitory effects of the whole lidocaine molecule on nAChRs.This work was supported by grants BFU2012-31359, SAF2015-66275-C2-1-R, BFU2011-25920, BFU2015-66612-P, and CSD2008-00005 from the MINECO and PROMETEO/2014/11 from GVA (Spain)