T-type voltage-gated calcium (Ca2+) channels play critical roles in controlling neuronal excitability, firing patterns, and synaptic plasticity, although the mechanisms and extent to which T-type Ca2+ channels are modulated by G-protein coupled receptors (GPCRs) remains largely unexplored. Investigations into T-type modulation within native neuronal systems have been complicated by the presence of multiple GPCR subtypes and a lack of pharmacological tools to separate currents generated by the three T-type isoforms; Cav3.1, Cav3.2, and Cav3.3. We hypothesize that specific Cav3 subtypes play unique roles in neuronal physiology due to their differential functional coupling to specific GPCRs. Co-expression of T-type channel subtypes and GPCRs in a heterologous system allowed us to identify the specific interactions between muscarinic acetylcholine (mAChR) or metabotropic glutamate (mGluR) GPCRs and individual Cav3 isoforms. Perforated patch recordings demonstrated that activation of Galpha<q/11>-coupled GPCRs had a strong inhibitory effect on Cav3.3 T-type Ca2+ currents but either no effect or a stimulating effect on Cav3.1 and Cav3.2 peak current amplitudes. Further study of the inhibition of Cav3.3 channels by a specific Galpha<q/11>-coupled mAChR (M1) revealed that this reversible inhibition was associated with a concomitant increase in inactivation kinetics. Pharmacological and genetic experiments indicated that the M1 receptor-mediated inhibition of Cav3.3 occurs specifically through a Galpha<q/11> signaling pathway that interacts with two distinct regions of the Cav3.3 channel. As hypothesized, the potentiation of Cav3.1 channels by a Galpha<q/11>-coupled mGluR (mGluR1) initially characterized in the heterologous system was also observed in a native neuronal system: the cerebellar Purkinje cell (PC). In recordings on PCs within acute cerebellar slices, we demonstrated that the potentiation of Cav3.1 currents by mGluR1 activation is strongest near the threshold of T-type currents, enhancing the excitability of PCs. Ultrafast two-photon Ca2+ imaging demonstrated that the functional coupling between mGluR1 and T-type transients occurs within dendritic spines, where synaptic integration and plasticity occurs. A subset of these experiments utilized physiological synaptic activation and specific mGluR1 antagonists in wild-type and Cav3.1 knock-out mice to show that the mGluR1-mediated potentiation of Cav3.1 T-type currents may promote synapse-specific Ca2+ signaling in response to bursts of excitatory inputs.

Thesis (Ph. D.)--University of Virginia, 2007. / Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available via the Internet as viewed 10 July 2008.

T-type voltage-gated calcium (Ca2+) channels play critical roles in controlling neuronal excitability, firing patterns, and synaptic plasticity, although the mechanisms and extent to which T-type Ca2+ channels are modulated by G-protein coupled receptors (GPCRs) remains largely unexplored. Investigations into T-type modulation within native neuronal systems have been complicated by the presence of multiple GPCR subtypes and a lack of pharmacological tools to separate currents generated by the three T-type isoforms; Cav3.1, Cav3.2, and Cav3.3. We hypothesize that specific Cav3 subtypes play unique roles in neuronal physiology due to their differential functional coupling to specific GPCRs. Co-expression of T-type channel subtypes and GPCRs in a heterologous system allowed us to identify the specific interactions between muscarinic acetylcholine (mAChR) or metabotropic glutamate (mGluR) GPCRs and individual Cav3 isoforms. Perforated patch recordings demonstrated that activation of Galpha<q/11>-coupled GPCRs had a strong inhibitory effect on Cav3.3 T-type Ca2+ currents but either no effect or a stimulating effect on Cav3.1 and Cav3.2 peak current amplitudes. Further study of the inhibition of Cav3.3 channels by a specific Galpha<q/11>-coupled mAChR (M1) revealed that this reversible inhibition was associated with a concomitant increase in inactivation kinetics. Pharmacological and genetic experiments indicated that the M1 receptor-mediated inhibition of Cav3.3 occurs specifically through a Galpha<q/11> signaling pathway that interacts with two distinct regions of the Cav3.3 channel. As hypothesized, the potentiation of Cav3.1 channels by a Galpha<q/11>-coupled mGluR (mGluR1) initially characterized in the heterologous system was also observed in a native neuronal system: the cerebellar Purkinje cell (PC). In recordings on PCs within acute cerebellar slices, we demonstrated that the potentiation of Cav3.1 currents by mGluR1 activation is strongest near the threshold of T-type currents, enhancing the excitability of PCs. Ultrafast two-photon Ca2+ imaging demonstrated that the functional coupling between mGluR1 and T-type transients occurs within dendritic spines, where synaptic integration and plasticity occurs. A subset of these experiments utilized physiological synaptic activation and specific mGluR1 antagonists in wild-type and Cav3.1 knock-out mice to show that the mGluR1-mediated potentiation of Cav3.1 T-type currents may promote synapse-specific Ca2+ signaling in response to bursts of excitatory inputs. / Medicine, Faculty of / Graduate

The aim of this thesis is to design a step-up DC/AC converter, an inverter from 12 V to 120 Vrms, with a sinus output signal. The converter should deliver a continuous performance of 300 W and a double peak power output of 600 W. The supposed usage of this inverter would be as a back-up power source for the circulatory pump of the central heating in the case of power outage. The inverter is consisting of a T-type power section.

Background: The Wnt/β-catenin signalling pathway is activated in arrhythmogenic heart diseases such as myocardial infarction and heart failure, but it is unclear if the pathway regulates cardiac ion channels and thus may play a role in arrhythmogenesis. Previous PCR array screening from our lab showed that the transcript level of the T-type calcium channel gene Cacna1g was reduced in primary culture of neonatal rat ventricular myocytes (NRVMs) after activation of Wnt/β-catenin signalling with Wnt3a protein (100 ng/ml) or a small molecule activator of the pathway, CHIR (3 µM) (n=3, p<0.01). In this study, we examined the effects of Wnt/β-catenin signalling on T-type calcium channels (Caᴠ3.1), which play a key role in the pacemaker function of the sinoatrial node (SAN). Results: RT-qPCR and western blot demonstrated dose-dependent reductions in Cacna1g mRNA (n=7, p<0.01) and Cav3.1 protein (n=4, p<0.01) in NRVMs after treatment with CHIR (3 μM). There was also a decrease in Cacna1g mRNA in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) after treatment with CHIR (5 μM) (n=4; p<0.001). Patch-clamp recording demonstrated reduced T-type calcium current (ICa,T) in NRVMs after Wnt3a treatment (3 μg/ml) (n=5, p<0.05). In isolated mouse SAN tissue, perfusion with an ICa,T blocker, ML-218 (30 µM), led to dose-dependent reductions in spontaneous beating rate (n=4, p<0.0001) indicating a critical role of ICa,T in SAN pacemaking. In adult rats, activation of Wnt/β-catenin signalling through the application of CHIR in a poloxamer gel to the SAN region did not alter the in vivo heart rate in electrocardiogram (ECG) (n=8, p=0.12). However, ex vivo culture of SAN tissue from the in vivo experiments revealed a reduction intrinsic beating rate in the CHIR treated group (n=7) compared to the control (DMSO) (n=8) (p<0.05). Summary: Wnt/β-catenin signalling inhibits T-type Ca²⁺ current in cardiomyocytes by, at least partly, reduced Cacna1g mRNA and Cav3.1 protein. Activation of Wnt/β-catenin signalling reduces the intrinsic heart rate likely by inhibition of T-type Ca²⁺ current in SAN pacemaker cells.

No description available.

No description available.

Cardiovascular disease remains the number one cause or mortally in the western world. Heart failure is the most rapidly growing cardiovascular disease (Hobbs, 2004; Levy, et al., 2002). Heart failure, by definition, is progressive deteriorating function of the heart due to progressive cardiac myocytes loss. Though after decades of endeavor of searching the pathophysiology and treatments for heart failure, it remains highly lethal. Therefore, it is vital to find novel therapies to help treat such chronic disease. Replace the lost cardiomyocyte with new ones could restore cardiac function and reduce mortality. The purpose of this study is to investigate on how TTCCs (T-type calcium channels) affect cardiomyocyte proliferation. In mice after birth, the major TTCC expressed in the heart is Cav3.1/α1G, and therefore we used Cav3.1/α1G transgenic (TG), knockout (-/-) and wild type mice respectively to define the role of TTCC in cardiomyocyte proliferation. In neonatal mouse ventricular myocyte (NMVMs) right after birth, there is almost no TTCC after birth in α1G-/- NMVMs, whereas there are around 35% NMVMs in wild type (WT) show TTCC. On day 7 after birth, there are no T-type calcium currents in both α1G-/- NMVMs and WT NMVMs. Using BrdU, a DNA synthesis marker, we identified plenty of BrdU positive cardiomyocyte during the first seven days after birth. Cardiomyocyte is special due to its double nucleation property. Our cell cycle studies showed that there is significant difference in cell cycle distribution between α1G-/- and WT NMVMs on day seven after birth. Significantly more NMVMs are arrested in G1 phase in α1G-/-, compared to WT NMVMs. Even until 2 month old, there are still significantly more mono-nucleated cardiomyocyte in α1G-/- than in WT. In conclusion, all these evidence showed that blocking T-type calcium channel could partially prevent binucleation from happening and stop cardiomyocytes withdrawal from cell cycle. Mononucleated cardiomyocyte is still able to proliferate. Hence, mononucleated cardiomyocytes in adult still have potential to proliferation because these cardiomyoctes are arrested in their cell-cycle before their terminal differentiation, which could offer a novel approach for cardiac repair. / Physiology

Yes / Ion channels represent a large and growing family of target proteins regulated by gasotransmitters such as nitric oxide, carbon monoxide and, as described more recently, hydrogen sulfide. Indeed, many of the biological actions of these gases can be accounted for by their ability to modulate ion channel activity. Here, we report recent evidence that H2S is a modulator of low voltage-activated T-type Ca2+ channels, and discriminates between the different subtypes of T-type Ca2+ channel in that it selectively modulates Cav3.2, whilst Cav3.1 and Cav3.3 are unaffected. At high concentrations, H2S augments Cav3.2 currents, an observation which has led to the suggestion that H2S exerts its pro-nociceptive effects via this channel, since Cav3.2 plays a central role in sensory nerve excitability. However, at more physiological concentrations, H2S is seen to inhibit Cav3.2. This inhibitory action requires the presence of the redox-sensitive, extracellular region of the channel which is responsible for tonic metal ion binding and which particularly distinguishes this channel isoform from Cav3.1 and 3.3. Further studies indicate that H2S may act in a novel manner to alter channel activity by potentiating the zinc sensitivity/affinity of this binding site. This review discusses the different reports of H2S modulation of T-type Ca2+ channels, and how such varying effects may impact on nociception given the role of this channel in sensory activity. This subject remains controversial, and future studies are required before the impact of T-type Ca2+ channel modulation by H2S might be exploited as a novel approach to pain management. / This work was supported by grants from the British Heart Foundation, the Medical Research Council, and the Hebei Medical University