Calcium currents via low-voltage-activated T-type channels mediate burst firing, particularly in thalamic neurons. Considerable evidence supports the hypothesis that overactive T-channels may contribute to thalamocortical dysrhythmia, including absence epilepsy. Single nucleotide polymorphisms in one of the T-channel genes (CACNA1H, which encodes Ca v 3.2) are associated with childhood absence epilepsy in a Chinese population. Because only a fraction of these polymorphisms are predicted to increase channel activity and neuronal firing, we hypothesized that other channel properties may be affected. Here we describe that all the polymorphisms clustered in the intracellular loop connecting repeats I and II (I-II loop) increase the surface expression of extracellularly tagged Ca v 3.2 channels. The functional domains within the I-II loop were then mapped by deletion analysis. The first 62 amino acids of the loop (post IS6) are involved in regulating the voltage dependence of channel gating and inactivation. Similarly, the last 15 amino acids of the loop (pre IIS1) are involved in channel inactivation. In contrast, the central region of I-II loop regulates surface expression, with no significant effect on channel biophysics. Electrophysiology, luminometry, fluorescence-activated cell sorting measurements, and confocal microscopy studies demonstrate that deletion of this central region leads to enhanced surface expression of channels from intracellular compartments to the plasma membrane. These results provide novel insights into how CACNA1H polymorphisms may contribute to Ca V 3.2 channel overactivity and consequently to absence epilepsy and establish the I-II loop as an important regulator of Ca V 3.2 channel function and expression.

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The mechanisms underlying cardiac automaticity are still incompletely understood and controversial. Here we report the complete conditional and time-controlled silencing of the "funny" current (If) by expression of a dominant-negative, non-conductive HCN4-channel subunit (hHCN4-AYA). Heart-specific If silencing caused altered [Ca2+]i release and Ca2+ handling in the sinoatrial node, impaired pacemaker activity, and symptoms reminiscent of severe human disease of pacemaking. The effects of If silencing critically depended on the activity of the autonomic nervous system. We were able to rescue the failure of impulse generation and conduction by additional genetic deletion of cardiac muscarinic G-protein-activated (GIRK4) channels in If-deficient mice without impairing heartbeat regulation. Our study establishes the role of f-channels in cardiac automaticity and indicates that arrhythmia related to HCN loss-of-function may be managed by pharmacological or genetic inhibition of GIRK4 channels, thus offering a new therapeutic strategy for the treatment of heart rhythm diseases.

Dysfunction of pacemaker activity in the sinoatrial node (SAN) underlies “sick sinus” syndrome (SSS), a common clinical condition characterized by abnormally low heart rate (bradycardia). If untreated, SSS carries potentially life-threatening symptoms, such as syncope and end-stage organ hypoperfusion. The only currently available therapy for SSS consists of electronic pacemaker implantation. Mice lacking L-type Cav1.3 Ca2+ channels (Cav1.3−/−) recapitulate several symptoms of SSS in humans, including bradycardia and atrioventricular (AV) dysfunction (heart block). Here, we tested whether genetic ablation or pharmacological inhibition of the muscarinic-gated K+ channel (IKACh) could rescue SSS and heart block in Cav1.3−/− mice. We found that genetic inactivation of IKACh abolished SSS symptoms in Cav1.3−/− mice without reducing the relative degree of heart rate regulation. Rescuing of SAN and AV dysfunction could be obtained also by pharmacological inhibition of IKACh either in Cav1.3−/− mice or following selective inhibition of Cav1.3-mediated L-type Ca2+ (ICa,L) current in vivo. Ablation of IKACh prevented dysfunction of SAN pacemaker activity by allowing net inward current to flow during the diastolic depolarization phase under cholinergic activation. Our data suggest that patients affected by SSS and heart block may benefit from IKACh suppression achieved by gene therapy or selective pharmacological inhibition.

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The intracellular loops that interlink the four transmembrane domains of Ca2+- and Na+-channels (Cav, Nav) have critical roles in numerous forms of channel regulation. In particular, the intracellular loop that joins repeats I and II (I–II loop) in high voltage-activated (HVA) Ca2+ channels possesses the binding site for Cavβ subunits and plays significant roles in channel function, including trafficking the α1 subunits of HVA channels to the plasma membrane and channel gating. Although there is considerable divergence in the primary sequence of the I–II loop of Cav1/Cav2 HVA channels and Cav3 LVA/T-type channels, evidence for a regulatory role of the I–II loop in T-channel function has recently emerged for Cav3.2 channels. In order to provide a comprehensive view of the role this intracellular region may play in the gating and surface expression in Cav3 channels, we have performed a structure-function analysis of the I–II loop in Cav3.1 and Cav3.3 channels using selective deletion mutants. Here we show the first 60 amino acids of the loop (post IS6) are involved in Cav3.1 and Cav3.3 channel gating and kinetics, which establishes a conserved property of this locus for all Cav3 channels. In contrast to findings in Cav3.2, deletion of the central region of the I–II loop in Cav3.1 and Cav3.3 yielded a modest increase (+30%) and a reduction (−30%) in current density and surface expression, respectively. These experiments enrich our understanding of the structural determinants involved in Cav3 function by highlighting the unique role played by the intracellular I–II loop in Cav3.2 channel trafficking, and illustrating the prominent role of the gating brake in setting the slow and distinctive slow activation kinetics of Cav3.3.

Modulation of low voltage-activated Ca V 3 T-type calcium channels remains poorly characterized compared with high voltage-activated Ca V 1 and Ca V 2 calcium channels. Notably, it is yet unresolved whether Ca V 3 channels are modulated by protein kinases in mammalian cells. In this study, we demonstrate that protein kinase A (PKA) and PKC (but not PKG) activation induces a potent increase in Ca V 3.1, Ca V 3.2, and Ca V 3.3 currents in various mammalian cell lines. Notably, we show that protein kinase effects occur at physiological temperature (ϳ30 -37°C) but not at room temperature (ϳ22-27°C). This temperature dependence could involve kinase translocation, which is impaired at room temperature. A similar temperature dependence was observed for PKC-mediated increase in high voltage-activated Ca V 2.3 currents. We also report that neither Ca V 3 surface expression nor T-current macroscopic properties are modified upon kinase activation. In addition, we provide evidence for the direct phosphorylation of Ca V 3.2 channels by PKA in in vitro assays. Overall, our results clearly establish the role of PKA and PKC in the modulation of Ca V 3 T-channels and further highlight the key role of the physiological temperature in the effects described.Voltage-gated Ca 2ϩ channels (VGCCs) 5 are unique among voltage-gated ion channels because the permeant Ca 2ϩ ion also acts as an intracellular second messenger, triggering diverse cellular functions (1, 2). VGCCs are therefore implicated in neuronal and cardiac excitability as well as in muscle contraction, neurotransmitter release or hormone secretion, and gene expression (1-6). Thus, the modulation of VGCCs plays a pivotal role in the control of cardiac and brain activities.VGCCs are divided into three families: the L-type channels (Ca V 1 family); the neuronal N-, P/Q-, and R-type channels (Ca V 2 family); and the T-type channels (Ca V 3 family) (7). Although the molecular mechanisms implicated in the modulation of high voltage-activated Ca 2ϩ channels of the Ca V 1 and Ca V 2 families are beginning to be unraveled (mainly, protein kinases for Ca V 1 channels and ␤␥-subunits of G proteins and protein kinase C (PKC) for Ca V 2 channels) (6, 8), those implicated in low voltage-activated Ca V 3 T-type channel regulation remain debated (9). Some transduction pathways mediate either decreases or increases in native T-currents, depending on the tissues/species studied and/or on the recording conditions (9). This apparent complexity could be explained by the existence of three T-channels (Ca V 3.1 or ␣ 1G , Ca V 3.2 or ␣ 1H , and Ca V 3.3 or ␣ 1I ), which include different splice variant isoforms with specific tissue patterns and developmental expression (10). Additional regulatory subunits of T-channels might also be involved in their modulation, but the native composition of T-channels remains unknown (10). Therefore, molecular studies on recombinant T-channels are required to clarify their modulation.Recent studies on recombinant T-channels have highlighted the complexity of the...

T-type calcium channels (T-channels) contribute to a wide variety of physiological functions, especially in the cardiovascular and nervous systems. Recent studies using knock-out mouse models have been instrumental in documenting further the role of T-channels in sleep, heartbeat, pain and epilepsy. Importantly, several novel aspects of the regulation of these channels have been identified over the last few years, providing new insights into their physiological and pathophysiological roles. Here, we review recent evidence supporting that the Cav3 subunits of T-channels are modulated by endogenous ligands such as anandamide, zinc, redox and oxidizing agents, as well as G-protein and protein kinases pathways. The study of T-channel mutations associated with childhood absence epilepsy has also revealed new aspects of Cav3 subunit trafficking. Collectively, these findings identify novel regulatory mechanisms involved in the fine tuning of T-channel expression and activity, and offer new directions for the design of novel therapeutic strategies targeting these channels.