Crucial points within the pore as determinants of K⁺ channel conductance and gating

While selective for K⁺, K⁺ channels vary significantly among their rate of ion permeation. Here, we probe the effect of steric hindrance and electrostatics within the ion conduction pathway on K⁺ permeation in the MthK K⁺ channel using structure-based mutagenesis combined with single-channel electrophysiology and X-ray crystallography. We demonstrate that changes in side-chain size and polarity at Ala88, which forms the constriction point of the open MthK pore, have profound effects on single-channel conductance as well as open probability. We also reveal that the negatively charged Glu92s at the intracellular entrance of the open pore form an electrostatic trap, which stabilizes a hydrated K⁺ and facilitates ion permeation. This electrostatic attraction is also responsible for intracellular divalent blockage, which renders the channel inward rectified in the presence of Ca²⁺. In light of the high structural conservation of the selectivity filter, the size and chemical environment differences within the portion of the ion conduction pathway other than the filter are likely the determinants for the conductance variations among K⁺ channels.     

天麻素通过下调Nav1.6通道表达缓解糖尿病神经病理性疼痛的研究

目的:探究天麻素对Ⅱ型糖尿病神经病理性痛的镇痛作用以及天麻素对背根神经节Nav1.6通道的表达调控作用。方法:将60只雄性SD大鼠随机分为空白对照组、糖尿病组和天麻素处理组(10 mg·kg^-1·d^-1)。通过高脂饮食喂养4周,低剂量腹腔注射STZ(30 mg·kg^-1)的方法构建Ⅱ型糖尿病神经病理性痛大鼠模型,利用痛行为学检测观察各组大鼠的机械刺激足缩反应阈值变化,采用免疫荧光组织化学及Western blot方法观察各组大鼠背根神经节上Nav1.6通道的表达变化。结果:与空白对照组相比,糖尿病模型大鼠出现显著的机械刺激疼痛阈值下降(P<0.05),且模型组大鼠背根神经节神经元上的Nav1.6通道表达上调(P<0.05)。与糖尿病组相比,连续腹腔注射天麻素3天、7天、14天后,模型动物的疼痛明显缓解(P<0.05),另外天麻素可以翻转背根神经节上Nav1.6通道的高表达(P<0.05)。结论:天麻素可能通过降低Nav1.6通道的表达来缓解Ⅱ型糖尿病神经病理性疼痛,从而为天麻素缓解糖尿病神经病理性疼痛提供新的理论依据。     

Functional and structural impact of pirfenidone on the alterations of cardiac disease and diabetes mellitus

A synthetic compound, termed pirfenidone (PFD), is considered promising for the treatment of cardiac disease. It leads to beneficial effects in animal models of diabetes mellitus (DM); as well as in heart attack, atrial fibrillation, muscular dystrophy, and diabetic cardiomyopathy (DC). The latter is a result of alterations linked to metabolic syndrome as they promote cardiac hypertrophy, fibrosis and contractile dysfunction. Although reduced level of fibrosis and stiffness represent an essential step in the mechanism of PFD action, a wide range of functional effects might also contribute to the therapeutic benefits. For example, PFD stimulates L-type voltage-gated Ca²⁺ channels (LTCCs), which are pivotal for a process known as excitation–contraction coupling (ECC). Recent evidence suggests that these two types of actions – namely structural and functional – aid in treating both cardiac disease and DM. This view is supported by the fact that in DC, for example, systolic dysfunction arises from both cardiac stiffness linked to fibrosis and down-regulation of ECC. Thus, not surprisingly, clinical trials have been conducted with PFD in the settings of DM, for treating not only cardiac but also renal disease. This review presents all these concepts, along with the possible mechanisms and pathophysiological consequences.     

Potential role of subunit c of F0F1-ATPase and subunit c of storage body in the mitochondrial permeability transition. Effect of the phosphorylation status of subunit c on pore opening

Phosphorylated and non-phosphorylated forms of the F₀F₁-ATPase subunit c from rat liver mitochondria (RLM) were purified and their effect on the opening of the permeability transition pore (mPTP) was investigated. Addition of dephosphorylated subunit c to RLM induced mitochondrial swelling, decreased the membrane potential and reduced the Ca²⁺ uptake capacity, which was prevented by cyclosporin A. The same effect was observed in the presence of storage subunit c purified from livers of sheep affected with ceroid lipofuscinosis. In black-lipid bilayer membranes subunit c increased the conductance due to formation of single channels with fast and slow kinetics. The dephosphorylated subunit c formed channels with slow kinetics, i.e. the open state being of significantly longer duration than in the case of channels formed by the phosphorylated form that had short life spans and fast kinetics. The channels formed were cation-selective more so with the phosphorylated form. Subunit c of rat liver mitochondria was able to bind Ca²⁺. Collectively, the data allowed us to suppose that subunit c F₀F₁-ATPase might be a structural/regulatory component of mPTP exerting its role in dependence on phosphorylation status.     

The effects of calcium channel blockade on proliferation and differentiation of cardiac progenitor cells

Cardiogenesis depends on a tightly regulated balance between proliferation and differentiation of cardiac progenitor cells (CPCs) and their cardiomyocyte descendants. While exposure of early mouse embryos to Ca²⁺ channel antagonists has been associated with abnormal cardiac morphogenesis, less is known about the consequences of Ca²⁺ channel blockade on proliferation and differentiation of CPCs at the cellular level. Here we showed that at embryonic day (E) 11.5, the murine ventricles express several L-type and T-type Ca²⁺ channel isoforms, and that the dihydropyridine Ca²⁺ channel antagonist, nifedipine, blunts isoproterenol induced increases in intracellular Ca²⁺. Nifedipine mediated Ca²⁺ channel blockade was associated with a reduction in cell cycle activity of E11.5 CPCs and impaired assembly of the cardiomyocyte contractile apparatus. Furthermore, in cell transplantation experiments, systemic administration of nifedipine to adult mice receiving transplanted E11.5 ventricular cells (containing CPCs and cardiomyocytes) was associated with smaller graft sizes compared to vehicle treated control animals. These data suggest that intracellular Ca²⁺ is a critical regulator of the balance between CPC proliferation and differentiation and demonstrate that interactions between pharmacological drugs and transplanted cells could have a significant impact on the effectiveness of cell based therapies for myocardial repair.     

At the right distance: ER-mitochondria juxtaposition in cell life and death

The interface between mitochondria and the endoplasmic reticulum is emerging as a crucial hub for calcium signalling, apoptosis, autophagy and lipid biosynthesis, with far reaching implications in cell life and death and in the regulation of mitochondrial and endoplasmic reticulum function. Here we review our current knowledge on the structural and functional aspects of this interorganellar juxtaposition. This article is part of a Special Issue entitled: Calcium Signaling In Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

Exploring emergent properties in cellular homeostasis using OnGuard to model K and other ion transport in guard cells

It is widely recognized that the nature and characteristics of transport across eukaryotic membranes are so complex as to defy intuitive understanding. In these circumstances, quantitative mathematical modeling is an essential tool, both to integrate detailed knowledge of individual transporters and to extract the properties emergent from their interactions. As the first, fully integrated and quantitative modeling environment for the study of ion transport dynamics in a plant cell, OnGuard offers a unique tool for exploring homeostatic properties emerging from the interactions of ion transport, both at the plasma membrane and tonoplast in the guard cell. OnGuard has already yielded detail sufficient to guide phenotypic and mutational studies, and it represents a key step toward ‘reverse engineering’ of stomatal guard cell physiology, based on rational design and testing in simulation, to improve water use efficiency and carbon assimilation. Its construction from the HoTSig libraries enables translation of the software to other cell types, including growing root hairs and pollen. The problems inherent to transport are nonetheless challenging, and are compounded for those unfamiliar with conceptual ‘mindset’ of the modeler. Here we set out guidelines for the use of OnGuard and outline a standardized approach that will enable users to advance quickly to its application both in the classroom and laboratory. We also highlight the uncanny and emergent property of OnGuard models to reproduce the ‘communication’ evident between the plasma membrane and tonoplast of the guard cell.     

Developmental refinement of hair cell synapses tightens the coupling of Ca2+ influx to exocytosis

Cochlear inner hair cells (IHCs) develop from pre‐sensory pacemaker to sound transducer. Here, we report that this involves changes in structure and function of the ribbon synapses between IHCs and spiral ganglion neurons (SGNs) around hearing onset in mice. As synapses matured they changed from holding several small presynaptic active zones (AZs) and apposed postsynaptic densities (PSDs) to one large AZ/PSD complex per SGN bouton. After the onset of hearing (i) IHCs had fewer and larger ribbons; (ii) Ca_V1.3 channels formed stripe‐like clusters rather than the smaller and round clusters at immature AZs; (iii) extrasynaptic Ca_V1.3‐channels were selectively reduced, (iv) the intrinsic Ca²⁺ dependence of fast exocytosis probed by Ca²⁺ uncaging remained unchanged but (v) the apparent Ca²⁺ dependence of exocytosis linearized, when assessed by progressive dihydropyridine block of Ca²⁺ influx. Biophysical modeling of exocytosis at mature and immature AZ topographies suggests that Ca²⁺ influx through an individual channel dominates the [Ca²⁺] driving exocytosis at each mature release site. We conclude that IHC synapses undergo major developmental refinements, resulting in tighter spatial coupling between Ca²⁺ influx and exocytosis.     

Rebuilding a macromolecular membrane complex at the atomic scale: Case of the Kir6.2 potassium channel coupled to the muscarinic acetylcholine receptor M2

Ion channel‐coupled receptors (ICCR) are artificial proteins built from a G protein‐coupled receptor and an ion channel. Their use as molecular biosensors is promising in diagnosis and high‐throughput drug screening. The concept of ICCR was initially validated with the combination of the muscarinic receptor M2 with the inwardly rectifying potassium channel Kir6.2. A long protein engineering phase has led to the biochemical characterization of the M2‐Kir6.2 construct. However, its molecular mechanism remains to be elucidated. In particular, it is important to determine how the activation of M2 by its agonist acetylcholine triggers the modulation of the Kir6.2 channel via the M2‐Kir6.2 linkage. In the present study, we have developed and validated a computational approach to rebuild models of the M2‐Kir6.2 chimera from the molecular structure of M2 and Kir6.2. The protocol was first validated on the known protein complexes of the μ‐opioid Receptor, the CXCR4 receptor and the Kv1.2 potassium channel. When applied to M2‐Kir6.2, our protocol produced two possible models corresponding to two different orientations of M2. Both models highlights the role of the M2 helices I and VIII in the interaction with Kir6.2, as well as the role of the Kir6.2 N‐terminus in the channel opening. Those two hypotheses will be explored in a future experimental study of the M2‐Kir6.2 construct. Proteins 2014; 82:1694–1707. © 2014 Wiley Periodicals, Inc.