Felix Neumaier

Voltage-gated calcium channels: Determinants of channel function and modulation by inorganic cations.

Voltage-gated calcium channels (VGCCs) represent a key link between electrical signals and non-electrical processes, such as contraction, secretion and transcription. Evolved to achieve high rates of Ca(2+)-selective flux, they possess an elaborate mechanism for selection of Ca(2+) over foreign ions. It has been convincingly linked to competitive binding in the pore, but the fundamental question of how this is reconcilable with high rates of Ca(2+) transfer remains unanswered. By virtue of their similarity to Ca(2+), polyvalent cations can interfere with the function of VGCCs and have proven instrumental in probing the mechanisms underlying selective permeation. Recent emergence of crystallographic data on a set of Ca(2+)-selective model channels provides a structural framework for permeation in VGCCs, and warrants a reconsideration of their diverse modulation by polyvalent cations, which can be roughly separated into three general mechanisms: (I) long-range interactions with charged regions on the surface, affecting the local potential sensed by the channel or influencing voltage-sensor movement by repulsive forces (electrostatic effects), (II) short-range interactions with sites in the ion-conducting pathway, leading to physical obstruction of the channel (pore block), and in some cases (III) short-range interactions with extracellular binding sites, leading to non-electrostatic modifications of channel gating (allosteric effects). These effects, together with the underlying molecular modifications, provide valuable insights into the function of VGCCs, and have important physiological and pathophysiological implications. Allosteric suppression of some of the pore-forming Cavα1-subunits (Cav2.3, Cav3.2) by Zn(2+) and Cu(2+) may play a major role for the regulation of excitability by endogenous transition metal ions. The fact that these ions can often traverse VGCCs can contribute to the detrimental intracellular accumulation of metal ions following excessive release of endogenous Cu(2+) and Zn(2+) or exposure to non-physiological toxic metal ions.

Cardiac phenomena during kainic-acid induced epilepsy and lamotrigine antiepileptic therapy

RATIONALE Pathologic ECG events are known to accompany seizures and to persist in several chronic epilepsy syndromes. The contribution of antiepileptic drugs (AEDs) to these events and the implications in the etiology of sudden-unexpected death in epilepsy (SUDEP) continue to be a matter of debate. We therefore investigated cardiac parameters during kainic-acid (KA) induced experimental epilepsy and antiepileptic treatment with lamotrigine (LTG). METHODS Epilepsy was induced in seven C57Bl/6 mice by injections of KA (20 mg/kg) on days 1 and 5, which produced severe acute seizures and spontaneous seizures 10 days later. Treatment with LTG (30 mg/kg) was initiated on day 11 and repeated on day 12. Continuous ECGs and ECoGs were collected telemetrically from freely moving mice. RESULTS Mice displayed pre-ictal but not ictal tachycardia. The squared coefficient of variation (SCV) of R-R intervals was significantly elevated 30s before and during seizures compared to control conditions. LTG produced a significant reversible increase in SCV and LF/HF ratio during slow-wave sleep (SWS), potentially indicative of sympatho-vagal imbalance during this state of vigilance, in which epileptic patients are known to be particularly vulnerable to SUDEP. SIGNIFICANCE The KA model used in this study permits the investigation of cardiac phenomena during epilepsy, as it features many effects found in human epileptic patients. Increased LF/HF, a known risk factor for cardiac disease, which is often found in epileptic patients, was observed as a side-effect of LTG treatment during SWS, suggesting that LTG may promote imbalance of the autonomous nervous system in epileptic mice.

Surgical Approaches in Psychiatry: A Survey of the World Literature on Psychosurgery

Brain surgery to promote behavioral or affective changes in humans remains one of the most controversial topics at the interface of medicine, psychiatry, neuroscience, and bioethics. Rapid expansion of neuropsychiatric deep brain stimulation has recently revived the field and careful appraisal of its 2 sides is warranted: namely, the promise to help severely devastated patients on the one hand and the dangers of premature application without appropriate justification on the other. Here, we reconstruct the vivid history of the field and examine its present status to delineate the progression from crude freehand operations into a multidisciplinary treatment of last resort. This goal is accomplished by a detailed reassessment of numerous case reports and small-scale open or controlled trials in their historical and social context. The different surgical approaches, their rationale, and their scientific merit are discussed in a manner comprehensible to readers lacking extensive knowledge of neurosurgery or psychiatry, yet with sufficient documentation to provide a useful resource for practitioners in the field and those wishing to pursue the topic further.

Electroretinographic Assessment of Inner Retinal Signaling in the Isolated and Superfused Murine Retina

ABSTRACT Purpose: Longer-lasting electroretinographic recordings of the isolated murine retina were initially achieved by modification of a phosphate-buffered nutrient solution originally developed for the bovine retina. During experiments with a more sensitive mouse retina, apparent model-specific limitations were addressed and improvements were analyzed for their contribution to an optimized full electroretinogram (ERG). Material and methods: Retinas were isolated from dark-adapted mice, transferred to a recording chamber and superfused with different solutions. Scotopic and photopic ERGs were recorded with white flashes every 3 minutes. The phosphate buffer (Sickel-medium) originally used was replaced by a carbonate-based system (Ames-medium), the pH of which was adjusted to 7.7–7.8. Moreover, addition of 0.1 mM BaCl2 was investigated to reduce b-wave contamination by the slow PIII component typically present in the murine ERG. Results: B-wave amplitudes were increased by the pH-shift (pH 7.4 to pH 7.7) from 22.9 ± 1.9 µV to 37.5 ± 2.5 µV. Improved b-wave responses were also achieved by adding small amounts of Ba2+ (100 µM), which selectively suppressed slow PIII components, thereby unmasking more of the true b-wave amplitude (100.0% with vs. 22.2 ± 10.7% without Ba2+). Ames medium lacking amino acids and vitamins was unable to maintain retinal signaling, as evident in a reversible decrease of the b-wave to 31.8 ± 3.9% of its amplitude in complete Ames medium. Conclusions: Our findings provide optimized conditions for ex vivo ERGs from the murine retina and suggest that careful application of Ba2+ supports reliable isolation of b-wave responses in mice. Under our recording conditions, murine retinas show reproducible ERGs for up to six hours.

Diethyldithiocarbamate-mediated zinc ion chelation reveals role of Cav2.3 channels in glucagon secretion.

Peptide-hormone secretion is partially triggered by Ca2+ influx through voltage-gated Ca2+ channels (VGCCs) and gene inactivation of Zn2+-sensitive Cav2.3-type VGCCs is associated with disturbed glucose homeostasis in mice. Zn2+ has been implicated in pancreatic islet cell crosstalk and recent findings indicate that sudden cessation of Zn2+ supply during hypoglycemia triggers glucagon secretion in rodents. Here we show that diethyldithiocarbamate (DEDTC), a chelating agent for Zn2+ and other group IIB metal ions, differentially affects blood glucose and serum peptide hormone level in wild-type mice and mice lacking the Cav2.3-subunit. Fasting glucose and glucagon level were significantly higher in Cav2.3-deficient compared to wild-type mice, while DEDTC Zn2+-chelation produced a significant and correlated increase of blood glucose and serum glucagon concentration in wild-type but not Cav2.3-deficient mice. Glucose tolerance tests revealed severe glucose intolerance in Zn2+-depleted Cav2.3-deficient but not vehicle-treated Cav2.3-deficient or Zn2+-depleted wildtype mice. Collectively, these findings indicate that Cav2.3 channels are critically involved in the Zn2+-mediated suppression of glucagon secretion during hyperglycemia. Especially under conditions of Zn2+ deficiency, ablation or dysfunction of Cav2.3 channels may lead to severe disturbances in glucose homeostasis.

Protein phosphorylation maintains the normal function of cloned human Cav2.3 channels

R-type currents mediated by native and recombinant Cav2.3 voltage-gated Ca2+ channels (VGCCs) exhibit facilitation (run-up) and subsequent decline (run-down) in whole-cell patch-clamp recordings. A better understanding of the two processes could provide insight into constitutive modulation of the channels in intact cells, but low expression levels and the need for pharmacological isolation have prevented investigations in native systems. Here, to circumvent these limitations, we use conventional and perforated-patch-clamp recordings in a recombinant expression system, which allows us to study the effects of cell dialysis in a reproducible manner. We show that the decline of currents carried by human Cav2.3+&bgr;3 channel subunits during run-down is related to adenosine triphosphate (ATP) depletion, which reduces the number of functional channels and leads to a progressive shift of voltage-dependent gating to more negative potentials. Both effects can be counteracted by hydrolysable ATP, whose protective action is almost completely prevented by inhibition of serine/threonine but not tyrosine or lipid kinases. Protein kinase inhibition also mimics the effects of run-down in intact cells, reduces the peak current density, and hyperpolarizes the voltage dependence of gating. Together, our findings indicate that ATP promotes phosphorylation of either the channel or an associated protein, whereas dephosphorylation during cell dialysis results in run-down. These data also distinguish the effects of ATP on Cav2.3 channels from those on other VGCCs because neither direct nucleotide binding nor PIP2 synthesis is required for protection from run-down. We conclude that protein phosphorylation is required for Cav2.3 channel function and could directly influence the normal features of current carried by these channels. Curiously, some of our findings also point to a role for leupeptin-sensitive proteases in run-up and possibly ATP protection from run-down. As such, the present study provides a reliable baseline for further studies on Cav2.3 channel regulation by protein kinases, phosphatases, and possibly proteases.

Reciprocal modulation of Cav2.3 voltage-gated calcium channels by copper(II) ions and kainic acid

Kainic acid (KA) is a potent agonist at non‐N‐methyl‐D‐aspartate (non‐NMDA) ionotropic glutamate receptors and commonly used to induce seizures and excitotoxicity in animal models of human temporal lobe epilepsy. Among other factors, Cav2.3 voltage‐gated calcium channels have been implicated in the pathogenesis of KA‐induced seizures. At physiologically relevant concentrations, endogenous trace metal ions (Cu2+, Zn2+) occupy an allosteric binding site on the domain I gating module of these channels and interfere with voltage‐dependent gating. Using whole‐cell patch‐clamp recordings in human embryonic kidney (HEK‐293) cells stably transfected with human Cav2.3d and β3‐subunits, we identified a novel, glutamate receptor‐independent mechanism by which KA can potently sensitize these channels. Our findings demonstrate that KA releases these channels from the tonic inhibition exerted by low nanomolar concentrations of Cu2+ and produces a hyperpolarizing shift in channel voltage‐dependence by about 10 mV, thereby reconciling the effects of Cu2+ chelation with tricine. When tricine was used as a surrogate to study the receptor‐independent action of KA in electroretinographic recordings from the isolated bovine retina, it selectively suppressed a late b‐wave component, which we have previously shown to be enhanced by genetic or pharmacological ablation of Cav2.3 channels. Although the pathophysiological relevance remains to be firmly established, we speculate that reversal of Cu2+‐induced allosteric suppression, presumably via formation of stable kainate‐Cu2+ complexes, could contribute to the receptor‐mediated excitatory effects of KA. In addition, we discuss experimental implications for the use of KA in vitro, with particular emphasis on the seemingly high incidence of trace metal contamination in common physiological solutions.

Unconjugated bilirubin modulates neuronal signaling only in wild-type mice, but not after ablation of the R-type/Cav2.3 voltage-gated calcium channel

The relationship between blood metabolites and hemoglobin degradation products (BMHDPs) formed in the cerebrospinal fluid and the development of vasospasm and delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (aSAH) has been the focus of several previous studies, but their molecular and cellular targets remain to be elucidated.

Multiple Nickel-Sensitive Targets Elicit Cardiac Arrhythmia in Isolated Mouse Hearts after Pituitary Adenylate Cyclase-Activating Polypeptide-Mediated Chronotropy.

Graphical abstract Figure. No Caption available. Abstract The pituitary adenylate cyclase‐activating polypeptide (PACAP)‐27 modulates various biological processes, from the cellular level to function specification. However, the cardiac actions of this neuropeptide are still under intense studies. Using control (+|+) and mice lacking (−|−) either R‐type (Cav2.3) or T‐type (Cav3.2) Ca2+ channels, we investigated the effects of PACAP‐27 on cardiac activity of spontaneously beating isolated perfused hearts. Superfusion of PACAP‐27 (20 nM) caused a significant increase of baseline heart frequency in Cav2.3(+|+) (156.9 ± 10.8 to 239.4 ± 23.4 bpm; p < 0.01) and Cav2.3(−|−) (190.3 ± 26.4 to 270.5 ± 25.8 bpm; p < 0.05) hearts. For Cav3.2, the heart rate was significantly increased in Cav3.2(−|−) (133.1 ± 8.5 bpm to 204.6 ± 27.9 bpm; p < 0.05) compared to Cav3.2(+|+) hearts (185.7 ± 11.2 bpm to 209.3 ± 22.7 bpm). While the P wave duration and QTc interval were significantly increased in Cav2.3(+|+) and Cav2.3(−|−) hearts following PACAP‐27 superfusion, there was no effect in Cav3.2(+|+) and Cav3.2(−|−) hearts. The positive chronotropic effects observed in the four study groups, as well as the effect on P wave duration and QTc interval were abolished in the presence of Ni2+ (50 &mgr;M) and PACAP‐27 (20 nM) in hearts from Cav2.3(+|+) and Cav2.3(−|−) mice. In addition to suppressing PACAP’s response, Ni2+ also induced conduction disturbances in investigated hearts. In conclusion, the most Ni2+‐sensitive Ca2+ channels (R‐ and T‐type) may modulate the PACAP signaling cascade during cardiac excitation in isolated mouse hearts, albeit to a lesser extent than other Ni2+‐sensitive targets.

A practical guide to the preparation and use of metal ion‐buffered systems for physiological research

Recent recognition that mobile pools of Zn2+ and Cu2+ are involved in the regulation of neuronal, endocrine and other cells has stimulated the development of tools to visualize and quantify the level of free trace metal ions. Most of the methods used to measure or control loosely bound metals require reference media that contain exactly defined free concentrations of the target ions. Despite the central importance of proper metal ion buffering, there is still a lack of international standards and beginners in the field may have difficulties finding a coherent description of how to prepare trace metal ion buffers, especially when experiments are to be performed in multimetal systems. To close this gap, we provide a guide for the design, preparation and use of metal ion‐buffered systems that facilitate immediate application under physiologically relevant ionic conditions. Thermodynamic and kinetic concepts of chemical speciation as well as general protocols and specific examples are outlined for the accurate preparation of single‐ and dual‐metal ion buffers. In addition, experiments have been performed with FluoZin‐3 to illustrate that metal ion‐buffered systems are required for reliable preparation of nanomolar Zn2+ solutions and that dual‐metal ion buffers can be used to calibrate suitable fluorescent Zn2+ sensors in the presence of millimolar Ca2+ concentrations. Together, the information provided should sensitize readers to the many potential pitfalls and uncertainties that exist when working with physiologically relevant concentrations of trace metal ions and enable them to formulate their own metal ion buffers for most in vitro applications.