A repulsion mechanism explains magnesium permeation and selectivity in CorA

Magnesium (Mg²⁺) plays a central role in biology, regulating the activity of many enzymes and stabilizing the structure of key macromolecules. In bacteria, CorA is the primary source of Mg²⁺ uptake and is self-regulated by intracellular Mg²⁺. Using a gating mutant at the divalent ion binding site, we were able to characterize CorA selectivity and permeation properties to both monovalent and divalent cations under perfused two-electrode voltage clamp. The present data demonstrate that under physiological conditions, CorA is a multioccupancy Mg²⁺-selective channel, fully excluding monovalent cations, and Ca²⁺, whereas in absence of Mg²⁺, CorA is essentially nonselective, displaying only mild preference against other divalents (Ca²⁺ > Mn²⁺ > Co²⁺ > Mg²⁺ > Ni²⁺). Selectivity against monovalent cations takes place via Mg²⁺ binding at a high-affinity site, formed by the Gly-Met-Asn signature sequence (Gly312 and Asn314) at the extracellular side of the pore. This mechanism is reminiscent of repulsion models proposed for Ca²⁺ channel selectivity despite differences in sequence and overall structure.Among biological divalent cations, Mg²⁺ is not only the most abundant, but also plays an essential role in a wealth of cellular processes, including enzymatic reactions, and the stability of nucleic acids and biological membranes (1). Although the biological importance of Mg²⁺ is well established, the molecular entities and mechanisms that govern its cellular homeostasis are not well understood. In bacteria, Mg²⁺ influx is primarily catalyzed by members of the CorA family of divalent ion transport systems (2, 3). The X-ray structure of CorA has provided an excellent template toward a molecular understanding of the mechanisms underlying Mg²⁺ influx (4–7). However, although CorA has been crystallized in a wide range of conditions, so far all available CorA structures seem to correspond to nonconductive conformations, which obviously limits the basic mechanistic insights regarding Mg²⁺ selectivity and translocation that can be derived from these high-resolution structures. Computational analyses, together with NMR, X-ray absorption, and Raman spectroscopy studies, have established that Mg²⁺ holds to its first hydration shell much more tightly than any other physiological cation (8–11); this implies that any Mg²⁺-selective transport system must either compensate for the high hydration energy (and accommodate the invariable octahedral geometry of this hexacoordinated ion) or establish a selectivity mechanism able to discriminate a hydrated or partially hydrated Mg²⁺ ion from monovalent and other divalent cations.Several hypotheses have been postulated to explain CorA’s function, including its role as a Mg²⁺ -selective channel (12), a Co²⁺ transporter (13), and even as an exporter of divalent cations (14). However, detailed mechanistic evaluation of CorA’s functional properties has been limited by the resolution of existing functional assays (15). Mg²⁺ transport through CorA depends on the combination of three parameters: (i) number of open gates, (ii) the electrical potential across the membrane, and (iii) the Mg²⁺ driving force, none of which can be properly controlled with sufficient time-resolution in in vivo experiments. Although a prokaryotic membrane protein, we have been able to heterologously express CorA in Xenopus oocytes, which, in combination with standard electrophysiological approaches, allowed us to measure CorA-catalyzed divalent macroscopic currents under a variety of ionic conditions. Crystallographic studies have suggested that intracellular Mg²⁺ act as the main regulator of CorA gating under physiological conditions (6). That Mg²⁺ acts as both a gating ligand and charge carrier ultimately complicates functional studies of CorA permeation and selectivity properties. To circumvent this issue we used a mutation at the divalent cation sensor that abolishes CorA Mg²⁺-dependent gating (Fig. 1A). This construct is ideally suited to evaluate ion permeation because it stabilizes steady-state currents by inhibiting the divalent ion-driven negative-feedback loop that defines CorA gating. Our results demonstrate that CorA is a bona fide multioccupancy ion channel, and that its divalent cation permeation and tight selectivity against monovalent cations can be explained on the basis of a block and repulsion mechanism, where the canonical “signature sequence” Gly-Met-Asn (GMN) plays a central role.Open in a separate windowFig. 1.CorA-driven Mg²⁺ currents recorded from TEVC. (A) The divalent cation sensor is highlighted on a cartoon representation of CorA crystal structure. Residues Asp89 and Asp253 are shown as purple sticks (B). The membrane potential (Vm) is clamped and held at −60 mV. The external solution is exchanged between two isosmotic buffers: one containing no monovalent or divalent cation (colored in gray on the horizontal bar), and one containing 20 mM Mg²⁺ (noted Mg²⁺). A representative trace recorded on a CorA–WT-expressing oocyte is shown in teal, and control oocyte trace is shown in gray and D253K in purple. The horizontal dotted line indicates the 0 A current level. (C) Representative traces of CorA D253K mutant in TEVC. The voltage pulse protocol is shown on top of the current traces. The dotted line represents the 0 current levels. (D) The corresponding I/V relationships recorded at different external Mg²⁺ concentrations are shown. The GHK-flux equation fits are displayed as solid lines, and experimental values are dots. (E) Mg²⁺ current recorded at −60 mV under external solution perfusion. The external solution is changed stepwise and the corresponding solution exchange protocol is superimposed to the trace. (F) Mean values (±SD) of several traces (n ≥ 5) were recorded and normalized to the maximum current. The values were plotted against the external [Mg²⁺] and fitted with a single-site binding curve.     

The nature of protein folding pathways

How do proteins fold, and why do they fold in that way? This Perspective integrates earlier and more recent advances over the 50-y history of the protein folding problem, emphasizing unambiguously clear structural information. Experimental results show that, contrary to prior belief, proteins are multistate rather than two-state objects. They are composed of separately cooperative foldon building blocks that can be seen to repeatedly unfold and refold as units even under native conditions. Similarly, foldons are lost as units when proteins are destabilized to produce partially unfolded equilibrium molten globules. In kinetic folding, the inherently cooperative nature of foldons predisposes the thermally driven amino acid-level search to form an initial foldon and subsequent foldons in later assisted searches. The small size of foldon units, ∼20 residues, resolves the Levinthal time-scale search problem. These microscopic-level search processes can be identified with the disordered multitrack search envisioned in the “new view” model for protein folding. Emergent macroscopic foldon–foldon interactions then collectively provide the structural guidance and free energy bias for the ordered addition of foldons in a stepwise pathway that sequentially builds the native protein. These conclusions reconcile the seemingly opposed new view and defined pathway models; the two models account for different stages of the protein folding process. Additionally, these observations answer the “how” and the “why” questions. The protein folding pathway depends on the same foldon units and foldon–foldon interactions that construct the native structure.     

Calcium entry into stereocilia drives adaptation of the mechanoelectrical transducer current of mammalian cochlear hair cells

Mechanotransduction in the auditory and vestibular systems depends on mechanosensitive ion channels in the stereociliary bundles that project from the apical surface of the sensory hair cells. In lower vertebrates, when the mechanoelectrical transducer (MET) channels are opened by movement of the bundle in the excitatory direction, Ca²⁺ entry through the open MET channels causes adaptation, rapidly reducing their open probability and resetting their operating range. It remains uncertain whether such Ca²⁺-dependent adaptation is also present in mammalian hair cells. Hair bundles of both outer and inner hair cells from mice were deflected by using sinewave or step mechanical stimuli applied using a piezo-driven fluid jet. We found that when cochlear hair cells were depolarized near the Ca²⁺ reversal potential or their hair bundles were exposed to the in vivo endolymphatic Ca²⁺ concentration (40 µM), all manifestations of adaptation, including the rapid decline of the MET current and the reduction of the available resting MET current, were abolished. MET channel adaptation was also reduced or removed when the intracellular Ca²⁺ buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) was increased from a concentration of 0.1 to 10 mM. The findings show that MET current adaptation in mouse auditory hair cells is modulated similarly by extracellular Ca²⁺, intracellular Ca²⁺ buffering, and membrane potential, by their common effect on intracellular free Ca²⁺.Hearing and balance depend on the transduction of mechanical stimuli into electrical signals. This process depends on the opening of mechanoelectrical transducer (MET) channels located at the tips of the shorter of pairs of adjacent stereocilia (1), which are specialized microvilli-like structures that form the hair bundles that project from the upper surface of hair cells (2,3). Deflection of hair bundles in the excitatory direction (i.e., toward the taller stereocilia) stretches specialized linkages, the tip-links, present between adjacent stereocilia (3–5), opening the MET channels. In hair cells from lower vertebrates, open MET channels reclose during constant stimuli via an initial fast adaptation mechanism followed by a much slower, myosin-based motor process, both of which are driven by Ca²⁺ entry through the channel itself (6–13). In mammalian auditory hair cells, MET current adaptation seems to be mainly driven by the fast mechanism (14–16), although the exact process by which it occurs is still largely unknown. The submillisecond speed associated with the adaptation kinetics of the MET channels in rat and mouse cochlear hair cells (17, 18) indicates that Ca²⁺, to cause adaptation, has to interact directly with a binding site on the channel or via an accessory protein (16). However, a recent investigation on rat auditory hair cells has challenged the view that Ca²⁺ entry is required for fast adaptation, and instead proposed an as-yet-undefined mechanism involving a Ca²⁺-independent reduction in the viscoelastic force of elements in series with the MET channels (19). In the present study, we further investigated the role of Ca²⁺ in MET channel adaptation in mouse cochlear hair cells by deflecting their hair bundles using a piezo-driven fluid jet, which is believed to produce a more uniform deflection of the hair bundles (20–23) compared with the piezo-driven glass rod (19, 24).     

Variations in radioiodine ablation: decision-making after total thyroidectomy

European Journal of Nuclear Medicine and Molecular Imaging - The role of radioiodine treatment following total thyroidectomy for differentiated thyroid cancer is changing. The last major revision...     

Impact of the type of anthracycline and of stem cell transplantation in younger patients with acute myeloid leukaemia: Long-term follow up of a phase III study

We provide a long-term evaluation of patients enrolled in the EORTC/GIMEMA AML-10 trial which included a total of 2157 patients, 15-60 years old, randomized to receive either daunorubicin (DNR, 50 mg/m²), mitoxantrone (MXR, 12 mg/m²), or idarubicin (IDA, 10 mg/m²) in addition to standard-dose cytarabine and etoposide for induction chemotherapy and intermediate dose cytarabine for consolidation. Younger patients who reached complete remission with complete (CR) or incomplete (CRi) recovery were then scheduled to receive an allogeneic hematopoietic stem cell transplantation (HSCT). That was if they had a HLA-identical sibling donor; in all other cases, an autologous HSCT had to be administered. At an 11-year median follow-up, the 5-year, 10-year and 15-year overall survival (OS) rates were 33.2%, 30.1% and 28.0%, respectively. No significant difference between the three randomized groups regarding OS was observed (P = .38). In young patients, 15-45 years old, no treatment difference (P = .89) regarding OS was observed, while in patients 46-60 years old, MXR and IDA groups had a trend for a longer OS as compared to the DNR group (P = .029). Among younger patients without a favorable MRC cytogenetic risk subgroup who achieved a CR/CRi after induction chemotherapy, those with a HLA-identical sibling donor had higher 10-year and 15-year OS rates than those without. In older patients who reached CR/CRi, the long-term outcomes of those with or without a donor was similar. In conclusion, long-term outcomes of the study confirmed similar OS in the three randomized groups in the whole cohort of patients.     

Cancer associated thrombosis in everyday practice: perspectives from GARFIELD-VTE

Journal of Thrombosis and Thrombolysis - Venous thromboembolism (VTE) is common in cancer patients and is an important cause of morbidity and mortality. The Global Anticoagulant Registry in the...     

Mukosa-Dreiecksknorpellappen nach Walter erweitert die innere Nasenklappe

Background

Reconstruction of the middle vault and intranasal valve abnormalities causing deformities and breathing problems still remain a challenge in rhinoplasty. In order to treat pathologies of this area, techniques such as the insertion of spreader grafts and butterfly grafts have been described so far.

Objectives

A broadening of the nasal breathing surface in the vault of the intranasal valve is not always achieved using these procedures. Are there any alternatives to the established techniques which lead to improved nasal breathing?

Methods

This article presents an alternative rhinoplasty technique by a closed approach for the correction of narrowed intranasal valves and the bony region of the piriform aperture. The surgical technique is described and representative cases are presented.

Conclusion

The correct application of the technique, a meticulous follow-up treatment and the wearing of a nasal endoepithesis for 3 months achieve a sustainable broadening of the intranasal valve with subjectively sufficient nasal breathing.     

Prise en charge des tumeurs neuroendocrines de l’intestin grêle

For neuroendocrine tumour of the small bowel treatment’s goals are to control the secretory syndrome and to cure the tumour. Carcinoid syndrome is usually due to metastatic spread to the liver. Control of this syndrome with somatostatin analogs is a priority. Even if there is metastatic spread, surgical treatment of the primitive tumour should be discussed in cases of retractile mesenteritis, small bowel ischemia or subocclusive syndrome in order to avoid any acute local complication. Surgical treatment of intrahepatic lesions is an option only if a complete resection of all lesions seems possible, and, if liver resection has to be large, an observation period is advisable. Embolisation is the best option if liver metastases are not resectable, with no extrahepatic lesions. Diffuse and non-evolving lesions should simply be monitored or treated with somatostatin analogs. Interferon, chemotherapy, peptide receptor radionuclide therapy should be proposed only in cases of demonstrated progressing lesions or uncontrolled carcinoid syndrome. Finally, it has to be emphasized that it is of the utmost importance to enrol those patients with very rare disease in prospective clinical trials currently open in France (Telestar, Evacel, Sunland, Netter-1).     

Gender Abuse,Depressive Symptoms,and Substance Use Among Transgender Women: A 3-Year Prospective Study

Objectives. We examined the effects of gender abuse (enacted stigma), depressive symptoms, and demographic, economic, and lifestyle factors on substance use among transgender women.Methods. We conducted a 3-year prospective study (December 2004 to September 2007) of 230 transgender women aged 19 to 59 years from the New York Metropolitan Area. Statistical techniques included generalized estimating equations with logistic and linear regression links.Results. Six-month prevalence of any substance use at baseline was 76.2%. Across assessment points, gender abuse was associated with alcohol, cannabis, cocaine, or any substance use during the previous 6 months, the number of days these substances were used during the previous month, and the number of substances used. Additional modeling associated changes in gender abuse with changes in substance use across time. Associations of gender abuse and substance use were mediated 55% by depressive symptoms. Positive associations of employment income, sex work, transgender identity, and hormone therapy with substance use were mediated 19% to 42% by gender abuse.Conclusions. Gender abuse, in conjunction with depressive symptoms, is a pervasive and moderately strong risk factor for substance use among transgender women. Improved substance abuse treatment is sorely needed for this population.Previous studies and reports have pointed to a high prevalence of substance use among transgender women.1,2 In surveys of this population in large US cities, self-reports of alcohol, cannabis, cocaine, amphetamine, methamphetamine, and opiate use have been 4 to 10 times as high as corresponding reports in the general population.3–6 A recent study of this population in the New York Metropolitan Area observed prevalence estimates of these substances that were, for the most part, marginally higher than previous reports (60.4% for heavy alcohol use, 40.0% for cannabis, 21.7% for cocaine, 3.9% for amphetamines and methamphetamines, and 3.5% for opiates).7Early clinical studies of this population attributed such high percentages of substance use to a gender identity at odds with sexual anatomy,8 with later reports emphasizing more socially based conflict described as “gender-variant living in an often hostile world.”9^(p88) Following minority stress theory,10 the use of alcohol and other drugs in lesbian, gay, bisexual, and transgender populations is now often understood as resulting from internalized stigma (including transgender phobia directed at oneself) or enacted stigma in the forms of discrimination or psychological or physical abuse by others.11–13Enacted stigma and substance use have been described in a few studies of lesbian, gay, bisexual, and transgender populations,14–16 but longitudinal investigations of these associations are rare,17 the findings have not been consistent,18 and no empirical research has focused on stigma and substance use among transgender women.19Recent prospective studies of transgender women by our research team have pointed to gender abuse (enacted stigma) as a pervasive risk factor for a range of interrelated adverse health outcomes. In one study, gender abuse was associated with incident HIV and sexually transmitted infection in part because of the mediating effect of depressive symptoms.20 A subsequent study showed moderately strong associations of psychological and physical gender abuse with incident major depression.21In this study, we furthered this line of inquiry by systematically examining gender abuse, depressive symptoms, and demographic, economic, and lifestyle variables as interrelated risk factors for substance use. We hypothesized that psychological and physical gender abuse (enacted stigma) would be associated with substance use across time. We also hypothesized that these associations would be partially mediated by depressive symptoms (i.e., gender abuse causes depression, which then causes substance use). We have observed associations of gender abuse and depression in our previous studies, and depression, in turn, has been linked to substance use in numerous clinical and population studies.22 One interpretation of the latter link, the self-medication hypothesis, suggests that depressed individuals use certain substances in an attempt to temporarily ameliorate their symptomatology.23Against the background of the previous study,21 which linked 4 background variables (employment income, sex work, social presentation of transgender identity, and hormone therapy) to depression in part because of the mediated effects of gender abuse, we hypothesized that these same background variables would likewise affect substance use in part because of the mediated effects of gender abuse. The link between employment income and gender abuse may reflect the social scrutiny of transgender women’s behavior in a formal workplace environment. Sex work (especially in public venues), social presentation of transgender identity, and physical feminization associated with hormone therapy may increase the public visibility of gender nonconformity and increase the odds of gender abuse as a result.