Modulation of Cav2.3 voltage-gated calcium channels by trace metal ions and trace metal chelators

BACKGROUND: The trace metal ions Zn2+ and Cu2+ are increasingly recognized as endogenous modulators of neuronal transmission, hormone secretion and synaptic plasticity. Cav2.3-type voltage-gated Ca2+ channels (VGCCs) are among their most sensitive targets and have an expression pattern that coincides with the spatial distribution of histochemically reactive trace metals in the brain, suggesting that they could represent a main mediator for their reported neuro-modulatory effects. Although non-conserved histidine residues on the external side of domain I have been convincingly implicated in the effects of trace metals on Cav2.3 channel gating, the exact mechanisms involved and their (patho)physiological relevance remain incompletely understood. AIMS: Aim of the articles compiled in the present thesis was to shed some light on the exact mechanisms of Zn2+- and Cu2+-induced Cav2.3 channel modulation and their potential relevance under normal and pathophysiological conditions. METHODS: In publication 1, crystallographic data of a Ca2+-selective bacterial model channel was used as a framework to theoretically analyze eukaryotic VGCC structure, function and modulation by inorganic cations. In publication 2, general protocols for preparation and use of metal ion-buffered solutions were developed and a fluorescent Zn2+ sensor was used to illustrate the importance of proper metal ion-buffering. In publication 3, conventional and perforated patch-clamp recordings together with different inhibitors and cytosolic factors were used to study Cav2.3 channel run-down during electrophysiological recordings, which was critical to optimize the conditions for experiments performed in publication 6. In publication 4, the effects of intraperitoneal injection of the Zn2+ chelator DEDTC on blood glucose homeostasis, glucose tolerance and peptide hormone secretion in Cav2.3-deficient and -competent mice were analyzed, insulin secretion was examined in isolated islets of Langerhans from both genotypes and the Zn2+-dependence of DEDTC effects on cloned Cav2.3 channels was verified using whole-cell patch-clamp recordings. In publication 5, whole-cell patch-clamp and electroretinographic recordings were used to characterize a receptor-independent but Cu2+-dependent mechanism of Cav2.3 channel modulation by the glutamate-receptor agonist kainic acid (KA). In publication 6, whole-cell patch-clamp recordings were used to characterize Zn2+-induced changes in Cav2.3 channel function and to develop a Markov model for Cav2.3 channel gating under control conditions and in the presence of physiological Zn2+ concentrations. RESULTS: Publication 1 provided novel insights into eukaryotic VGCC function and modulation by trace metal ions. Publication 2 demonstrated the critical importance of proper metal ion buffering to avoid deviations between nominal and actual free metal ion concentrations. Publication 3 showed that run-down of Cav2.3 channel currents is associated with changes in channel gating and that it can be prevented or delayed by hydrolysable ATP through a mechanism that critically depends on protein phosphorylation by serine/threonine kinases. Publication 4 revealed severe glucose intolerance in Zn2+-depleted Cav2.3-deficient but not vehicle-treated Cav2.3-deficient or Zn2+-depleted wildtype mice. In addition, fasting glucose and glucagon levels were significantly higher in Cav2.3-deficient mice, whereas Zn2+ chelation significantly increased blood glucose and glucagon concentrations in wildtype but not Cav2.3-deficient mice. Application of DEDTC significantly stimulated cloned human Cav2.3 channels when applied in the presence of Zn2+ but had no effect in the presence of the Zn2+ chelator CaEDTA. Publication 5 uncovered that KA can stimulate cloned human Cav2.3 channels in the absence of functional KA receptors by reversing Cu2+-induced suppression in vitro, presumably via formation of stable kainate-Cu2+ complexes. When the chelator tricine was used as a surrogate to study the receptor-independent effects of KA in the isolated bovine retina, it selectively reduced a late ERG b-wave component that was previously shown to be enhanced by pharmacologic or genetic ablation of Cav2.3 channels. Publication 6 demonstrated that Zn2+-induced changes in Cav2.3 channel function are complex and inconsistent with a single mechanisms of action. Computer simulations were used to show that most, but not all of the effects can be reconciled by a simplified Markov model that involves Zn2+ binding to a first site with an associated electrostatic modification and mechanical slowing of one of the voltage-sensors and Zn2+-binding to a second, lower affinity site which blocks the channel and modifies the opening and closing transitions. DISCUSSION: With regard to Zn2+-induced Cav2.3 channel modulation, the results in publication 6 point to an intricate dependence on the prevailing neuronal properties and ionic conditions, which could profoundly influence and even invert the net Zn2+ action. Thus, due to Zn2+-induced parallel changes in activation and inactivation voltage-dependence, the net action is strongly affected by the holding potential, can be either inhibitory or stimulatory and may persist for several minutes after cessation of the Zn2+ signal. This could conceivably play a role for certain forms of synaptic sensitization or plasticity, and might also be relevant for e.g. the regulation of Cav2.3 channels in pancreatic islets, where sudden cessation of Zn2+ supply from β-cells is thought to serve as one of the switch-off signals for α-cell glucagon secretion. In support of the latter notion, the findings in publication 4 provide evidence for an involvement of Cav2.3 channels in the Zn2+-mediated suppression of glucagon secretion during hyperglycemia and indicate that Cav2.3 channel dysfunction could lead to severe disturbances in glucose homeostasis, especially under conditions of Zn2+-deficiency. Based on the results of publications 5 and 6, a decrease or reversal of Zn2+ and Cu2+-induced Cav2.3 channel suppression by endogenous (i.e. glutamate) or exogenous (i.e. KA) trace metal chelators, moderate acidification or depolarization of the neuronal resting membrane potential could also contribute to the pro-convulsive role of Cav2.3 channels demonstrated in previous investigations, although the pathophysiological relevance of these finding in vivo remains to be firmly established. Finally, the findings in publication 3 suggest that protein phosphorylation is required for normal Cav2.3 channel function and that it could modify the normal properties of currents carried by these channels. Conclusion: The articles compiled in this thesis provide several novel insights into the mechanisms underlying reciprocal Cav2.3 channel modulation by trace metal ions and trace metal chelators as well as first evidence for their importance under (patho)physiological conditions. Moreover, while still far from complete, the model developed in publication 6 provides a quantitative framework for understanding Zn2+ effects on Cav2.3 channel function and a first step towards the application of computational approaches for predicting the complex action of Zn2+ on neuronal excitability

Die Benzolformel

In dieser Abhandlung wird die Geschichte der Benzolformel von der geschlossenen Kette (1865) und der Oszillationshypothese (1872) Kekulés bis zur quantenmechanischen Behandlung durch E. Hückel (1931) und L. Pauling (1933) nachgezeichnet. Die erstmalige Formulierung des Benzolmoleküls als Sechseck mit abwechselnd Doppel- und Einfachbindungen geht auf Claus (1866) zurück. Die heftig diskutierte Frage, ob Loschmidt bereits 1861 eine ringförmige Verkettung von sechs C-Atomen zur Wiedergabe des Benzolkerns entwickelt hat, wird sorgfältig untersucht. Die Gründe, die in der zweiten Hälfte des 19. Jahrhundert zur Aufstellung alternativer Formeln geführt haben, werden diskutiert: Diagonalformel (Claus 1866), Prismenformel (Claus 1866, Ladenburg 1869), Dewar-Formel (Städeler 1868, Dewar 1869), Zentrische Formel (L. Meyer 1872, Armstrong 1887, Baeyer 1887), Thiele-Formel (1899). Die zentrische und die Thiele-Formel kommen unserer heutigen, elektronentheoretisch begründeten Auffassung von der Natur des Benzols am nächsten. Eine Benzolformel, die das Molekül mit einer besonderen Packung von sechs C-Tetraedern beschreibt, geht auf Marsh (1882), Loschmidt (1890) und Erlenmeyer jun. (1901) zurück. Armstrong (1890), Bamberger (1893) und Crocker (1922) haben die Idee des aromatischen Sextetts entwickelt, allerdings ohne diesen Begriff einzuführen. Sie umschrieben das aromatische Sextett mit double ring (Hexagon mit hinein gesetztem “c“), hexazentrischen Valenzen und six aromatic electrons. Die endgültige Formulierung mit einem Kreis im Hexagon stammt von Robinson (1925). Später hat Clar (1972) mit seiner aromatischen Pi-Sextett-Regel ein Ordnungsprinzip für polycyclische aromatische Kohlenwasserstoffe vorgeschlagen. In den dreißiger Jahren des 20. Jahrhunderts entwickelten E. Hückel (1931) und Pauling (1933) die Valence-Bond- bzw. Resonanztheorie und erklärten damit die besonderen Eigenschaften des Benzols auf quantenmechanischer Grundlage. Das zweite quantenmechanische Verfahren, die Hückel-Molekülorbital-Methode (1931), zeigt, dass alle planaren Ringe mit 4n+2 Pi-Elektronen abgeschlossene, energetisch stabilisierte Elektronenkonfigurationen ausbilden. Die Weiterentwicklung der quantenmechanischen Verfahren hin zu den ab-initio-Methoden hat zu immer genaueren und detaillierteren Erkenntnissen der Bindungsverhältnisse des Benzolmoleküls geführt.This essay depicts the history of the benzene formula reaching from the closed chain (chaîne fermée 1865) and the oscillation hypothesis (1872) of Kekulé to the quantum mechanical treatment of E. Hückel (1931) and L. Pauling (1932). The first formulation of the benzene molecule as a hexagon with alternating double and single bonds can be found in a report written by Claus (1866). A graphical representation of benzene by Loschmidt (1861) awoke a heated controversy about the question whether this scientist was the first to describe the benzene nucleus as a cyclic linkage of six carbon atoms. This problem is carefully analysed. The reasons why several alternative benzene formulae were established in the second half of the 19th century are discussed: diagonal formula (Claus 1866), prism formula (Claus 1866, Ladenburg 1869), Dewar formula (Städeler 1868, Dewar 1869), centric formula (L. Meyer 1872, Armstrong 1887, Baeyer 1887), Thiele formula (1899). The centric and the Thiele formula reveal close similarities to our present understanding based on electronic theory. A benzene formula describing the molecule as a special packing of six carbon tetraeders was developed by Marsh (1882), Loschmidt (1890) and Erlenmeyer jun. (1901). Armstrong (1890), Bamberger (1893) and Crocker (1922) inaugurated the idea of the aromatic sextet, however, without using this term. Instead, they used the following paraphrases “double ring” (expressed by placing “c” for centric into the hexagon), “hexacentric valencies” and “six aromatic electrons”. In 1925 Robinson symbolized the concept of the aromatic sextet by drawing a circle into the benzene hexagon. Later, in 1972, Clar created on the basis of his “aromatic pi- sextet rule” a classification principle for polycyclic aromatic hydrocarbons. In the 1930s E. Hückel (1931) and L. Pauling (1933) developed the valence bond or resonance theory to explain the particular properties and structure of benzene on a quantum theoretical base. The second quantum theoretical procedure, the Hückel molecular orbital theory (1931) provided the cognition that, in general, all planar rings having 4n+2 pi-electons possess stable closed-shell electronic configurations. The progress of the quantum theoretical procedures (ab initio methods) yielded increasingly precise and detailed insights into the bonding conditions of the benzene molecule

Nuclear Medicine in Times of COVID-19: How Radiopharmaceuticals Could Help to Fight the Current and Future Pandemics

The emergence and global spread of COVID-19, an infectious disease caused by the novel coronavirus SARS-CoV-2, has resulted in a continuing pandemic threat to global health. Nuclear medicine techniques can be used for functional imaging of (patho)physiological processes at the cellular or molecular level and for treatment approaches based on targeted delivery of therapeutic radionuclides. Ongoing development of radiolabeling methods has significantly improved the accessibility of radiopharmaceuticals for in vivo molecular imaging or targeted radionuclide therapy, but their use for biosafety threats such as SARS-CoV-2 is restricted by the contagious nature of these agents. Here, we highlight several potential uses of nuclear medicine in the context of SARS-CoV-2 and COVID-19, many of which could also be performed in laboratories without dedicated containment measures. In addition, we provide a broad overview of experimental or repurposed SARS-CoV-2-targeting drugs and describe how radiolabeled analogs of these compounds could facilitate antiviral drug development and translation to the clinic, reduce the incidence of late-stage failures and possibly provide the basis for radionuclide-based treatment strategies. Based on the continuing threat by emerging coronaviruses and other pathogens, it is anticipated that these applications of nuclear medicine will become a more important part of future antiviral drug development and treatment

Mutated Isocitrate Dehydrogenase (mIDH) as Target for PET Imaging in Gliomas

Gliomas are the most common primary brain tumors in adults. A diffuse infiltrative growth pattern and high resistance to therapy make them largely incurable, but there are significant differences in the prognosis of patients with different subtypes of glioma. Mutations in isocitrate dehydrogenase (IDH) have been recognized as an important biomarker for glioma classification and a potential therapeutic target. However, current clinical methods for detecting mutated IDH (mIDH) require invasive tissue sampling and cannot be used for follow-up examinations or longitudinal studies. PET imaging could be a promising approach for non-invasive assessment of the IDH status in gliomas, owing to the availability of various mIDH-selective inhibitors as potential leads for the development of PET tracers. In the present review, we summarize the rationale for the development of mIDH-selective PET probes, describe their potential applications beyond the assessment of the IDH status and highlight potential challenges that may complicate tracer development. In addition, we compile the major chemical classes of mIDH-selective inhibitors that have been described to date and briefly consider possible strategies for radiolabeling of the most promising candidates. Where available, we also summarize previous studies with radiolabeled analogs of mIDH inhibitors and assess their suitability for PET imaging in gliomas

Nuclear Medicine in Times of COVID-19: How Radiopharmaceuticals Could Help to Fight the Current and Future Pandemics

The emergence and global spread of COVID-19, an infectious disease caused by the novel coronavirus SARS-CoV-2, has resulted in a continuing pandemic threat to global health. Nuclear medicine techniques can be used for functional imaging of (patho)physiological processes at the cellular or molecular level and for treatment approaches based on targeted delivery of therapeutic radionuclides. Ongoing development of radiolabeling methods has significantly improved the accessibility of radiopharmaceuticals for in vivo molecular imaging or targeted radionuclide therapy, but their use for biosafety threats such as SARS-CoV-2 is restricted by the contagious nature of these agents. Here, we highlight several potential uses of nuclear medicine in the context of SARS-CoV-2 and COVID-19, many of which could also be performed in laboratories without dedicated containment measures. In addition, we provide a broad overview of experimental or repurposed SARS-CoV-2-targeting drugs and describe how radiolabeled analogs of these compounds could facilitate antiviral drug development and translation to the clinic, reduce the incidence of late-stage failures and possibly provide the basis for radionuclide-based treatment strategies. Based on the continuing threat by emerging coronaviruses and other pathogens, it is anticipated that these applications of nuclear medicine will become a more important part of future antiviral drug development and treatment

Drug Penetration into the Central Nervous System: Pharmacokinetic Concepts and In Vitro Model Systems

Delivery of most drugs into the central nervous system (CNS) is restricted by the blood–brain barrier (BBB), which remains a significant bottleneck for development of novel CNS-targeted therapeutics or molecular tracers for neuroimaging. Consistent failure to reliably predict drug efficiency based on single measures for the rate or extent of brain penetration has led to the emergence of a more holistic framework that integrates data from various in vivo, in situ and in vitro assays to obtain a comprehensive description of drug delivery to and distribution within the brain. Coupled with ongoing development of suitable in vitro BBB models, this integrated approach promises to reduce the incidence of costly late-stage failures in CNS drug development, and could help to overcome some of the technical, economic and ethical issues associated with in vivo studies in animal models. Here, we provide an overview of BBB structure and function in vivo, and a summary of the pharmacokinetic parameters that can be used to determine and predict the rate and extent of drug penetration into the brain. We also review different in vitro models with regard to their inherent shortcomings and potential usefulness for development of fast-acting drugs or neurotracers labeled with short-lived radionuclides. In this regard, a special focus has been set on those systems that are sufficiently well established to be used in laboratories without significant bioengineering expertise

Validation of analytical HPLC with post-column injection as a method for rapid and precise quantification of radiochemical yields

Accurate assessment of isolated radiochemical yields (RCYs) is a prerequisite for efficient and reliable optimization of labeling reactions. In practice, radiochemical conversions (RCCs) determined by HPLC analysis of crude reaction mixtures are often used to estimate RCYs. However, incomplete recovery of radioactivity from the stationary phase can lead to significant inaccuracies if RCCs are calculated based on the activity eluted from the column (i.e. the summed integrals of all peaks). Here, we validate a simple and practical method that overcomes problems associated with retention of activity on the column by determination of the total activity in the sample using post-column injection. Post-column injections were carried out using an additional injection valve, which was placed between the outlet of the HPLC column and the inlet of the detectors. 2-[18F]Fluoropyridine ([18F]FPy) and 8-cyclopentyl-3-(3-[18F]fluoropropyl)-1-propylxanthine ([18F]CPFPX) were prepared with radiochemical purities of >99.8% and mixed with [18F]fluoride at a ratio of 1:1 to simulate reaction mixtures obtained by radiolabeling reactions with an RCC of 50%. The samples were analyzed on three different C18 HPLC columns using neutral and acidic mobile phases. RCCs determined using the summed area of all peaks in the chromatograms were compared with those determined using post-column injection. Additionally, RCCs determined by post-column injection were corrected for activity losses before, during and after radiosyntheses to afford analytical RCYs, which were compared with isolated RCYs. Determination of RCCs based on the summed area of all peaks gave correct results under certain chromatographic conditions, but led to overestimation of the actual RCCs by up to 50% in other cases. In contrast, determination of RCCs using post-column injection provided precise results in all cases, and often significantly reduced analysis time. Moreover, analytical RCYs calculated from RCCs determined by post-column injection showed excellent agreement with isolated RCYs (<3% deviation). In conclusion, HPLC analysis using post-column injection enables reliable determination of RCCs independent of the chromatographic conditions and, together with a simple activity balance, rapid and accurate prediction of isolated RCYs

Ca(v)2.3 channel function and Zn2+-induced modulation: potential mechanisms and (patho)physiological relevance

Voltage-gated calcium channels (VGCCs) are critical for Ca(2+)influx into all types of excitable cells, but their exact function is still poorly understood. Recent reconstruction of homology models for all human VGCCs at atomic resolution provides the opportunity for a structure-based discussion of VGCC function and novel insights into the mechanisms underlying Ca(2+)selective flux through these channels. In the present review, we use these data as a basis to examine the structure, function, and Zn2+-induced modulation of Ca(v)2.3 VGCCs, which mediate native R-type currents and belong to the most enigmatic members of the family. Their unique sensitivity to Zn(2+)and the existence of multiple mechanisms of Zn(2+)action strongly argue for a role of these channels in the modulatory action of endogenous loosely bound Zn2+, pools of which have been detected in a number of neuronal, endocrine, and reproductive tissues. Following a description of the different mechanisms by which Zn(2+)has been shown or is thought to alter the function of these channels, we discuss their potential (patho)physiological relevance, taking into account what is known about the magnitude and function of extracellular Zn(2+)signals in different tissues. While still far from complete, the picture that emerges is one where Ca(v)2.3 channel expression parallels the occurrence of loosely bound Zn(2+)pools in different tissues and where these channels may serve to translate physiological Zn(2+)signals into changes of electrical activity and/or intracellular Ca(2+)levels