Electrophysiological characterization and interplay of TRPC1 channels and voltage-gated Ca2+ channels

In many non-excitable cells, which do not generate action potentials by themselves, extracellular chemical signals, like hormones and neurotransmitters, are transduced into membrane depolarizations, which in turn activate voltage-gated Ca2+ (CaV) channels. Hormones and their GPCRs activate transient receptor potential TRPC channels, which allow Na+ and Ca2+ influx, initiating Ca2+-dependent signalling pathways and, in addition, depolarize the plasma membrane and thereby activate CaV channels. To be able to investigate the interplay of TRPC and CaV channels the present study focused on the electrophysiological characterization of L- and T-type CaVs, their β subunits and TRPC ion channels, especially TRPC1, TRPC4, TRPC5, TRPC4/TRPC1 and TRPC5/TRPC1. Expression of TRPC1 wild-type or TRPC1 with mutations at the putative lower gate in HEK-cells did not reveal any detectable current. However, TRPC1 coexpressed with TRPC4 reveals functional heteromeric TRPC4/TRPC1 channels with altered biophysical properties as compared to monomeric TRPC4. While most of the TRPC1 mutations had no effect, TRPC1 V741A significantly increased TRPC4/TRPC1 currents. In addition, chimeras of TRPC4 backbone with TRPC1 pore revealed constitutive activity, sensitive to the TRPC blocker SKF 96365. The results suggest that TRPC1 contributes to the pore in TRPC4/TRPC1 heteromers and can form a permeable constitutive active channel pore itself. A subset of pituitary cells functionally expresses heteromeric TRPC5/TRPC1 channels. Pituitary cells isolated from TRPC1-deficient mice revealed homomeric TRPC5 currents with altered current-voltage relationship, lower amplitudes and higher Ca2+ permeability as compared to TRPC5/TRPC1 heteromeric channels in wild-type. Thus, TRPC1 modulates activity and Ca2+ permeability of TRPC5 in vivo and thereby the Ca2+ influx in pituitary cells. Auxiliary subunits, the CaVβ proteins, are required for the function of CaV channels. To study the impact of CaVβ subunits on the function of L- and T-type CaV channels, different β subunits were coexpressed with the CaVs in HEK-cells. CaVβ2 splice variants increased the L-type CaV1.2 current, but did not significantly affect T-type CaV3.2 currents, whereas CaVβ3 significantly inhibit CaV3.2 currents. In addition, CaVβ3 serves Ca2+ channel independent functions in fibroblasts, where I could not detect CaV currents and where CaVβ3 changes the sensitivity of the inositol trisphosphate (IP3) receptor for low concentrations of IP3. CaV3.2 currents are also inhibited by Englerin A (EA; EC50 222 nM), which had so far assumed to be a specific agonist of TRPC4, TRPC5, TRPC4/TRPC1 and TRPC5/TRPC1 channels. An electrophysiological characterization of a CaV3.2 mutant, identified in a Spanish family and linked to hyper-aldosteronism and hypertension is ongoing.In vielen nicht erregbaren Zellen, die selbst keine Aktionspotentiale erzeugen, werden extrazelluläre chemische Signale wie beispielsweise Hormone und Neurotransmitter in Membrandepolarisationen umgewandelt, die wiederum spannungsabhängige Calcium Kanäle aktivieren. Die Hormone beziehungsweise die durch sie stimulierten GPCRs können transiente Rezeptorpotential TRPC Kanäle aktivieren, was einen Na+- und Ca2+-Einstrom ermöglicht. Der Ca2+-Einstrom initiiert Ca2+-abhängige Signalwege, und der Na+-Einstrom führt zur Depolarisation der Plasmamembran wodurch spannungsgesteuerte Ca2+ (CaV) Kanäle aktiviert werden. Um das Zusammenspiel von TRPC und CaV Kanälen untersuchen zu können, konzentrierte sich die vorliegende Studie auf die elektrophysiologische Charakterisierung von CaVs vom L- und T-Typ, ihren β-Untereinheiten und von TRPC Ionenkanälen, insbesondere TRPC1, TRPC4, TRPC5, TRPC4/TRPC1 und TRPC5/TRPC1. Die Expression von TRPC1 Wildtyp oder TRPC1 mit Mutationen am mutmaßlichen unteren Gate ergab in HEK-Zellen keinen nachweisbaren Strom. Mit TRPC4 coexprimiert führt TRPC1 jedoch zu funktionellen heteromeren TRPC4/TRPC1 Kanälen, die veränderte biophysikalische Eigenschaften im Vergleich zu monomerem TRPC4 aufweisen. Während die meisten TRPC1 Mutationen keine Wirkung auf TRPC4/TRPC1 Ströme hatten, erhöhte TRPC1 V741A die TRPC4/TRPC1 Ströme signifikant. Zusätzlich zeigten Chimären des TRPC4-Rückgrats mit TRPC1-Poren eine konstitutive Aktivität, die mit dem TRPC-Blocker SKF 96365 inhibiert werden konnte. Die Ergebnisse legen nahe, dass TRPC1 zur Pore in TRPC4/TRPC1-heteromeren Kanälen beiträgt und selbst eine Ionenpermeable konstitutiv aktive Kanalpore bilden kann. Ein Teil von Hypophysenzellen exprimiert funktionell heteromere TRPC5/TRPC1 Kanäle. Aus TRPC1-defizienten Mäusen isolierte Hypophysenzellen zeigten homomere TRPC5 Ströme mit veränderter Strom-Spannungs-Beziehung, niedrigeren Amplituden und höherer Ca2+-Permeabilität im Vergleich zu heteromeren TRPC5/TRPC1 Kanälen in Wildtyp Hypophysenzellen. Somit moduliert TRPC1 die Aktivität und Ca2+-Permeabilität von TRPC5 in vivo und damit den Ca2+-Einstrom in die Hypophysenzellen. Die CaVβ-Proteine werden für die Funktion von CaV Kanälen benötigt. Um den Einfluss von CaVβ-Untereinheiten auf die Funktion von CaV Kanälen vom L- und T-Typ zu untersuchen, wurden verschiedene β-Untereinheiten mit den CaVs in HEK-Zellen coexprimiert. CaVβ2-Spleißvarianten erhöhten den CaV1.2 Strom vom L-Typ, beeinflussten jedoch die CaV3.2 Ströme vom T-Typ nicht signifikant, während CaVβ3 die CaV3.2 Ströme signifikant inhibierte. Darüber hinaus erfüllt CaVβ3 CaV-unabhängige Funktionen in Fibroblasten, bei denen ich keinen CaV Strom nachweisen Zusammenfassung XXV konnte und bei denen CaVβ3 die Empfindlichkeit des Inositoltrisphosphat (IP3) Rezeptors für niedrige IP3-Konzentrationen verändert. CaV3.2 Ströme werden auch durch Englerin A (EA, EC50 222 nM) gehemmt. Für EA wurde bisher angenommen, dass es ein spezifischer Agonist von TRPC4, TRPC5, TRPC4/TRPC1 und TRPC5/TRPC1 Kanälen ist. Die elektrophysiologische Charakterisierung einer CaV3.2 Mutante, die in einer spanischen Familie identifiziert und mit Hyperaldosteronismus und Hypertonie in Verbindung gebracht wurde, ist noch nicht abgeschlossen. Der Anhang enthält zusätzliche Experimente zu den im Ergebnissteil beschriebenen Daten

The Pathophysiology and Treatment of Essential Tremor: The Role of Adenosine and Dopamine Receptors in Animal Models

Essential tremor (ET) is one of the most common neurological disorders that often affects people in the prime of their lives, leading to a significant reduction in their quality of life, gradually making them unable to independently perform the simplest activities. Here we show that current ET pharmacotherapy often does not sufficiently alleviate disease symptoms and is completely ineffective in more than 30% of patients. At present, deep brain stimulation of the motor thalamus is the most effective ET treatment. However, like any brain surgery, it can cause many undesirable side effects; thus, it is only performed in patients with an advanced disease who are not responsive to drugs. Therefore, it seems extremely important to look for new strategies for treating ET. The purpose of this review is to summarize the current knowledge on the pathomechanism of ET based on studies in animal models of the disease, as well as to present and discuss the results of research available to date on various substances affecting dopamine (mainly D3) or adenosine A1 receptors, which, due to their ability to modulate harmaline-induced tremor, may provide the basis for the development of new potential therapies for ET in the future

IP3-dependent Ca2+ signals are tightly controlled by Cavβ3, but not by Cavβ1, 2 and 4

Independent of its function as a subunit of voltage-gated Ca2+ channels, the Cavβ3 subunit desensitizes fibroblasts and pancreatic β-cells to low concentrations of inositol-1,4,5-trisphosphate (IP3). This alters agonist-induced Ca2+ signaling and cellular functions, for example, insulin secretion and wound healing. A total of four Cavβ subunits exist, Cavβ1, Cavβ2, Cavβ3, and Cavβ4. To investigate whether the other Cavβ subunits, like Cavβ3, can desensitize cells to IP3 and thereby modulate Ca2+ signaling, we expressed the cDNAs of Cavβ1, Cavβ2, Cavβ3, and Cavβ4 in COS-7 cells lacking endogenous Cavβ proteins. ATP stimulation of these cells results in the release of Ca2+ from intracellular stores. This receptor-mediated Ca2+ release is significantly decreased by Cavβ3 but not by Cavβ1, Cavβ2, and Cavβ4. Electrophysiological recordings of voltage-dependent Ca2+ currents from fibroblasts show a small Ca2+ current, the amplitude of which is slightly but not significantly smaller in fibroblasts from Cavβ2 gene-deficient animals than in fibroblasts from wild-type animals. Compared with fibroblasts from wild-type animals, Ca2+ release is not significantly increased in Cavβ2-deficient fibroblasts, in contrast to Ca2+ release in Cavβ3-deficient fibroblasts. In summary, our results show that desensitization of cells to low concentrations of IP3 is a specific property of Cavβ3 that is not shared by other Cavβ subunits

The influence of 6-OHDA, pramipexole and imipramine on BDNF and trkB mRNAs in the caudate-putamen.

<p>n = 9–10. For further explanations see Figs. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117698#pone.0117698.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117698#pone.0117698.g002" target="_blank">2</a>.</p

Alterations of BDNF and trkB mRNA Expression in the 6-Hydroxydopamine-Induced Model of Preclinical Stages of Parkinson’s Disease: An Influence of Chronic Pramipexole in Rats

<div><p>Our recent study has indicated that a moderate lesion of the mesostriatal and mesolimbic pathways in rats, modelling preclinical stages of Parkinson’s disease, induces a depressive-like behaviour which is reversed by chronic treatment with pramipexole. The purpose of the present study was to examine the role of brain derived neurotrophic factor (BDNF) signalling in the aforementioned model of depression. Therefore, we investigated the influence of 6-hydoxydopamine (6-OHDA) administration into the ventral region of the caudate-putamen on mRNA levels of BDNF and tropomyosin-related kinase B (trkB) receptor. The BDNF and trkB mRNA levels were determined in the nigrostriatal and limbic structures by <i>in situ</i> hybridization 2 weeks after the operation. Pramipexole (1 mg/kg sc twice a day) and imipramine (10 mg/kg ip once a day) were injected for 2 weeks. The lesion lowered the BDNF and trkB mRNA levels in the hippocampus [CA1, CA3 and dentate gyrus (DG)] and amygdala (basolateral/lateral) as well as the BDNF mRNA content in the habenula (medial/lateral). The lesion did not influence BDNF and trkB expression in the caudate-putamen, substantia nigra, nucleus accumbens (shell and core) and ventral tegmental area (VTA). Chronic imipramine reversed the lesion-induced decreases in BDNF mRNA in the DG. Chronic pramipexole increased BDNF mRNA, but decreased trkB mRNA in the VTA in lesioned rats. Furthermore, it reduced BDNF and trkB mRNA expression in the shell and core of the nucleus accumbens, BDNF mRNA in the amygdala and trkB mRNA in the caudate-putamen in these animals. The present study indicates that both the 6-OHDA-induced dopaminergic lesion and chronic pramipexole influence BDNF signalling in limbic structures, which may be related to their pro-depressive and antidepressant activity in rats, respectively.</p></div

Representative autoradiograms showing BDNF and trkB mRNAs expression in frontal sections of the brain.

<p>Regions of interest are outlined. AMG—amygdala, CP—caudate-putamen, DG—dentate gyrus, HB—habenula, NAC—nucleus accumbens, SN—substantia nigra, VTA—ventral tegmental area. AP—anterior-posterior levels according to Paxinos and Watson [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117698#pone.0117698.ref045" target="_blank">45</a>].</p

The influence of 6-OHDA, pramipexole and imipramine on BDNF and trkB mRNAs in the habenula.

<p>n = 8–10. For further explanations see Figs. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117698#pone.0117698.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117698#pone.0117698.g002" target="_blank">2</a>.</p

IP3 Receptor-Dependent Cytoplasmic Ca2+ Signals Are Tightly Controlled by Cavβ3

Summary: Voltage-gated calcium channels (Cavs) are major Ca2+ entry pathways in excitable cells. Their β subunits facilitate membrane trafficking of the channel’s ion-conducting α1 pore and modulate its gating properties. We report that one β subunit, β3, reduces Ca2+ release following stimulation of phospholipase C-coupled receptors and inositol 1,4,5-trisphosphate (IP3) formation. This effect requires the SH3-HOOK domain of Cavβ3, includes physical β3/IP3 receptor interaction, and prevails when agonist-induced IP3 formation is bypassed by photolysis of caged IP3. In agreement with β3 acting as a brake on Ca2+ release, fibroblast migration is enhanced in vitro, and in vivo, closure of skin wounds is accelerated in the absence of β3. To mediate specific physiological responses and to prevent Ca2+ toxicity, cytoplasmic Ca2+ signals must be tightly controlled. The described function of β3, unrelated to its function as a Cav subunit, adds to this tight control. : Belkacemi et al. show that the calcium channel subunit Cavβ3 binds to the IP3R and desensitizes cells to low IP3 levels, influencing fibroblast migration and collagen secretion. Removal of the Cavβ3 protein in mice results in faster skin wound healing. Keywords: Ca2+ signaling, Cavβ3, cell migration, IP3 receptor, wound healing, Cavβ2, Cavβ3 KO, IP3 binding, voltage-gated Ca2+ channel, Ca2+ releas

The influence 6-OHDA, pramipexole and imipramine on BDNF and trkB mRNAs in the amygdala.

<p>n = 8–10. For further explanations see Figs. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117698#pone.0117698.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117698#pone.0117698.g002" target="_blank">2</a>.</p

The influence of 6-OHDA, pramipexole and imipramine on trkB mRNA in the hippocampus.

<p>n = 9–10. For further explanations see Figs. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117698#pone.0117698.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117698#pone.0117698.g002" target="_blank">2</a>.</p