Di-4-ANEPPS Modulates Electrical Activity and Progress of Myocardial Ischemia in Rabbit Isolated Heart.

AIMS: Although voltage-sensitive dye di-4-ANEPPS is a common tool for mapping cardiac electrical activity, reported effects on electrophysiological parameters are rather. The main goals of the study were to reveal effects of the dye on rabbit isolated heart and to verify, whether rabbit isolated heart stained with di-4-ANEPPS is a suitable tool for myocardial ischemia investigation. METHODS AND RESULTS: Study involved experiments on stained (n = 9) and non-stained (n = 11) Langendorff perfused rabbit isolated hearts. Electrophysiological effects of the dye were evaluated by analysis of various electrogram (EG) parameters using common paired and unpaired statistical tests. It was shown that staining the hearts with di-4-ANEPPS leads to only short-term sporadic prolongation of impulse conduction through atria and atrioventricular node. On the other hand, significant irreversible slowing of heart rate and ventricular conduction were found in stained hearts as compared to controls. In patch clamp experiments, significant inhibition of sodium current density was observed in differentiated NG108-15 cells stained by the dye. Although no significant differences in mean number of ventricular premature beats were found between the stained and the non-stained hearts in ischemia as well as in reperfusion, all abovementioned results indicate increased arrhythmogenicity. In isolated hearts during ischemia, prominent ischemic patterns appeared in the stained hearts with 3-4 min delay as compared to the non-stained ones. Moreover, the ischemic changes did not achieve the same magnitude as in controls even after 10 min of ischemia. It resulted in poor performance of ischemia detection by proposed EG parameters, as was quantified by receiver operating characteristics analysis. CONCLUSION: Our results demonstrate significant direct irreversible effect of di-4-ANEPPS on spontaneous heart rate and ventricular impulse conduction in rabbit isolated heart model. Particularly, this should be considered when di-4-ANEPPS is used in ischemia studies in rabbit. Delayed attenuated response of such hearts to ischemia might lead to misinterpretation of obtained results.

Grina/TMBIM3 modulates voltage-gated CaV2.2 Ca2+ channels in a G-protein-like manner.

Grina/TMBIM3 is a poorly characterized transmembrane protein with a broad expression pattern in mammals and with a very ancient origin within eukaryotes. Although initially characterized as an NMDA-receptor associated subunit, there is increasing evidence that Grina/TMBIM3 is involved in the unfolded protein response and controls apoptosis via regulation of Ca2+ homeostasis. Here, we investigate a putative direct interaction of Grina/TMBIM3 with voltage gated Ca2+ channels, in particular with the CaV2.2 α1-subunit and describe its modulatory effects on the current through CaV2.2 N-type channels. Direct interaction was confirmed by co-immunoprecipitation studies and membrane localization was proven. Co-expression of Grina/TMBIM3 with CaV2.2 channels resulted in a significant decrease of the current amplitude and in a slowing of the kinetics of current activation. This effect was accompanied by a significant shift of the voltage dependencies of activation time constants towards more depolarized voltages. Application of a stimulus protocol including a strong depolarizing pulse relieved inhibition of current amplitude by Grina/TMBIM3. When Grina/TMBIM3 was present, inactivation by an action potential-like train of pulses was diminished. Both observations resemble mechanisms that are well-studied modulatory effects of G-protein ßγ subunits on CaV2 channels. The impact of Grina/TMBIM3 and G-protein ßγ subunits are rather comparable with respect to suppression of current amplitude and slowing of activation kinetics. Furthermore, both modulators had the same effect on current inactivation when evoked by an action potential-like train of pulses.

Correction to: Four novel interaction partners demonstrate diverse modulatory effects on voltage-gated CaV2.2 Ca2+ channels.

The article was originally published with one author missing. The name of the co-author Roman Moravcik was inadvertently omitted. His name and affiliation have now been added to the author list. The original article has been corrected.

Four novel interaction partners demonstrate diverse modulatory effects on voltage-gated CaV2.2 Ca2+ channels.

Voltage-gated Ca2+ channels are embedded in a network of protein interactions that are fundamental for channel function and modulation. Different strategies such as high-resolution quantitative MS analyses and yeast-two hybrid screens have been used to uncover these Ca2+ channel nanodomains. We applied the yeast split-ubiquitin system with its specific advantages to search for interaction partners of the CaV2.2 Ca2+ channel and identified four proteins: reticulon 1 (RTN1), member 1 of solute carrier family 38 (SLC38), prostaglandin D2 synthase (PTGDS) and transmembrane protein 223 (TMEM223). Interactions were verified using the yeast split-ubiquitin system and narrowed down to CaV2.2 domain IV. Colocalization studies using fluorescent constructs demonstrated defined regions of subcellular localization. Detailed electrophysiological studies revealed that coexpression of RTN1 modulated CaV2.2 channels only to a minor extent. SLC38 accelerated the cumulative current inactivation during a high-frequency train of brief depolarizing pulses. As neurons expressing CaV2.2 channels were exposed to high-frequency bursts under physiological conditions, observed regulation may have a negative modulatory effect on transmitter release. Coexpression of PTGDS significantly lowered the average current density and slowed the kinetics of cumulative current inactivation. Since the latter effect was not significant, it may only partly compensate the first one under physiological conditions. Expression of TMEM223 lowered the average current density, accelerated the kinetics of cumulative current inactivation and slowed the kinetics of recovery from inactivation. Therefore, TMEM223 and, to a lesser extent, PTGDS, may negatively modulate Ca2+ entry required for transmitter release and/or for dendritic plasticity under physiological conditions.

Fibrotic scar model and TGF-ß1 differently modulate action potential firing and voltage-dependent ion currents in hippocampal neurons in primary culture.

Traumatic injury of the central nervous system is accompanied by various functional and morphological changes. Animal models of traumatic brain injury are commonly used to investigate changes in behaviour, morphology, in the expression of various proteins around the site of the injury, or the expression of diagnostically important biomarkers. Excitability of a single neuron at, or close to, the site of injury was rarely investigated. Several in vitro models were developed which allow such investigation. In the present work, we employed a fibrotic scar model according to Kimura-Kuroda and coauthors to analyse altered excitability of rat hippocampal neurons under the conditions mimicking traumatic brain injury. Hippocampal neurons from newborn rats were cultured either on a fibrotic scar model or in the presence of TGF-ß1, a cytokine secreted at a brain injury site that may have both neuroprotective and neurodegenerative function. Fibrotic scar facilitated ability of neonatal hippocampal neurons to fire action potential series by increasing the density of voltage activated sodium and potassium currents. Chondroitin sulphate proteoglycans played substantial role in these effects, as proven by their full reversion after administration of Chondroitinase ABC. In contrast, TGF-ß1 did not contribute to them. An application of TGF-ß1 itself attenuated generation of action potentials, inhibited sodium current and potentiated potassium currents. Main alteration of electrophysiological parameters of neonatal hippocampal neurons caused by a fibrotic scar model is enhanced excitability. TGF-ß1 may have predominantly neuroprotective role in injured rat hippocampus.

Altered Sodium and Potassium, but not Calcium Currents in Cerebellar Granule Cells in an In Vitro Model of Neuronal Injury.

Acute injury of central nervous system (CNS) starts a cascade of morphological, molecular, and functional changes including formation of a fibrotic scar, expression of transforming growth factor beta 1 (TGF-ß1), and expression of extracellular matrix proteins leading to arrested neurite outgrowth and failed regeneration. We assessed alteration of electrophysiological properties of cerebellar granule cells (CGCs) in two in vitro models of neuronal injury: (i) model of fibrotic scar created from coculture of meningeal fibroblasts and cerebral astrocytes with addition of TGF-ß1; (ii) a simplified model based on administration of TGF-ß1 to CGCs culture. Both models reproduced suppression of neurite outgrowth caused by neuronal injury, which was equally restored by chondroitinase ABC (ChABC), a key disruptor of fibrotic scar formation. Voltage-dependent calcium current was not affected in either injury model. However, intracellular calcium concentration could be altered as an expression of inositol trisphosphate receptor type 1 was suppressed by TGF-ß1 and restored by ChABC. Voltage-dependent sodium current was significantly suppressed in CGCs cultured on a model of fibrotic scar and was only partly restored by ChABC. Administration of TGF-ß1 significantly shifted current-voltage relation of sodium current toward more positive membrane potential without change to maximal current amplitude. Both transient and sustained potassium currents were significantly suppressed on a fibrotic scar and restored by ChABC to their control amplitudes. In contrast, TGF-ß1 itself significantly upregulated transient and did not change sustained potassium current. Observed changes of voltage-dependent ion currents may contribute to known morphological and functional changes in injured CNS.

Modulation of Cav3.2 T-type calcium channel permeability by asparagine-linked glycosylation.

Low-voltage-gated T-type calcium channels are expressed throughout the nervous system where they play an essential role in shaping neuronal excitability. Defects in T-type channel expression have been linked to various neuronal disorders including neuropathic pain and epilepsy. Currently, little is known about the cellular mechanisms controlling the expression and function of T-type channels. Asparagine-linked glycosylation has recently emerged as an essential signaling pathway by which the cellular environment can control expression of T-type channels. However, the role of N-glycans in the conducting function of T-type channels remains elusive. In the present study, we used human Cav3.2 glycosylation-deficient channels to assess the role of N-glycosylation on the gating of the channel. Patch-clamp recordings of gating currents revealed that N-glycans attached to hCav3.2 channels have a minimal effect on the functioning of the channel voltage-sensor. In contrast, N-glycosylation on specific asparagine residues may have an essential role in the conducting function of the channel by enhancing the channel permeability and / or the pore opening of the channel. Our data suggest that modulation of N-linked glycosylation of hCav3.2 channels may play an important physiological role, and could also support the alteration of T-type currents observed in disease states.