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Cav3.2 T-type calcium channels are important mediators of nociceptive signaling, but their roles in the transmission of itch remains poorly understood. Here we report a key involvement of these channels as key modulators of itch/pruritus-related behavior. We compared scratching behavior responses between wild type and Cav3.2 null mice in models of histamine- or chloroquine-induced itch. We also evaluated the effect of the T-type calcium channel blocker DX332 in male and female wild-type mice injected with either histamine or chloroquine. Cav3.2 null mice exhibited decreased scratching responses during both histamine- and chloroquine-induced acute itch. DX332 co-injected with the pruritogens inhibited scratching responses of male and female mice treated with either histamine or chloroquine. Altogether, our data provide strong evidence that Cav3.2 T-type channels exert an important role in modulating histamine-dependent and -independent itch transmission in the primary sensory afferent pathway, and highlight these channels as potential pharmacological targets to treat pruritus.

Caveolae position Ca V 3.2 (T-type Ca 2+ channel encoded by the α-3.2 subunit) sufficiently close to RyR (ryanodine receptors) for extracellular Ca 2+ influx to trigger Ca 2+ sparks and large-conductance Ca 2+ -activated K + channel feedback in vascular smooth muscle. We hypothesize that this mechanism of Ca 2+ spark generation is affected by age. Using smooth muscle cells (VSMCs) from mouse mesenteric arteries, we found that both Ca v 3.2 channel inhibition by Ni 2+ (50 µM) and caveolae disruption by methyl-ß-cyclodextrin or genetic abolition of Eps15 homology domain-containing protein (EHD2) inhibited Ca 2+ sparks in cells from young (4 months) but not old (12 months) mice. In accordance, expression of Ca v 3.2 channel was higher in mesenteric arteries from young than old mice. Similar effects were observed for caveolae density. Using SMAKO Ca v 1.2 -/- mice, caffeine (RyR activator) and thapsigargin (Ca 2+ transport ATPase inhibitor), we found that sufficient SR Ca 2+ load is a prerequisite for the Ca V 3.2-RyR axis to generate Ca 2+ sparks. We identified a fraction of Ca 2+ sparks in aged VSMCs, which is sensitive to the TRP channel blocker Gd 3+ (100 µM), but insensitive to Ca V 1.2 and Ca V 3.2 channel blockade. Our data demonstrate that the VSMC Ca V 3.2-RyR axis is down-regulated by aging. This defective Ca V 3.2-RyR coupling is counterbalanced by a Gd 3+ sensitive Ca 2+ pathway providing compensatory Ca 2+ influx for triggering Ca 2+ sparks in aged VSMCs., (© 2020 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

The deletion of T-type Ca v 3.1 channels may reduce high-fat diet (HFD)-induced weight gain, which correlates positively with obesity and endothelial dysfunction. Therefore, experiments were designed to study the involvement of T-type Ca v 3.1 channels in HFD-induced endothelial dysfunction in mice. Wildtype (WT) and Ca v 3.1 -/- mice were fed either a normal diet (ND) or an HFD for 8 weeks. Body composition was assessed, and thoracic aortae and mesenteric arteries were harvested for myography to assess endothelium-dependent responses. Changes in intracellular calcium were measured by fluorescence imaging, and behavior was assessed with the open-field test. Ca v 3.1 -/- mice had attenuated HFD-induced weight gain and lower total fat mass compared with WT mice. Ca v 3.1 -/- mice on an HFD had reduced plasma cholesterol levels compared with WT mice on the same diet. Increased feeding efficiency, independent of food intake, was observed in WT mice on an HFD compared with an ND, but no difference in feeding efficiency between diets was observed for Ca v 3.1 -/- mice. Nitric oxide-dependent dilatation was increased in mesenteric arteries of Ca v 3.1 -/- mice compared with WT mice on an HFD, with no difference observed in aortae. No differences in mouse locomotor activity were observed between the experimental groups. Mice on an HFD lacking T-type channels have reduced weight gain, lower total cholesterol levels, and increased dilatation of resistance vessels compared with WT mice on an HFD, suggesting that Ca v 3.1 deletion protects against endothelial dysfunction in resistance vessels but not in large conduit vessels.

The spontaneous contractions of collecting lymphatic vessels provide an essential propulsive force to return lymph centrally. These contractions are driven by an intrinsic electrical pacemaker, working through an unknown underlying ionic mechanism that becomes compromised in some forms of lymphedema. In previous studies, T-type voltage-gated Ca 2+ channels (VGCCs) were implicated in this pacemaking mechanism, based on the effects of the reputedly selective T-type VGCC inhibitors mibefradil and Ni 2+ . Our goal was to test this idea in a more definitive way using genetic knock out mice. First, we demonstrated through both PCR and immunostaining that mouse lymphatic muscle cells expressed Ca v 3.1 and Ca v 3.2 and produced functional T-type VGCC currents when patch clamped. We then employed genetic deletion strategies to selectively test the roles of each T-type VGCC isoform in the regulation of lymphatic pacemaking. Surprisingly, global deletion of either, or both, isoform(s) was without significant effect on either the frequency, amplitude, or fractional pump flow of lymphatic collectors from two different regions of the mouse, studied ex vivo. Further, both WT and Ca v 3.1 -/- ; 3.2 -/- double knock-out lymphatic vessels responded similarly to mibefradil and Ni 2+ , which substantially reduced contraction amplitudes and slightly increased frequencies at almost all pressures in both strains: a pattern consistent with inhibition of L-type rather than T-type VGCCs. Neither T-type VGCC isoform was required for ACh-induced inhibition of contraction, a mechanism by which those channels in smooth muscle are thought to be targets of endothelium-derived nitric oxide. Sharp intracellular electrode measurements in lymphatic smooth muscle revealed only subtle, but not significant, differences in the resting membrane potential and action potential characteristics between vessels from wild-type and Ca v 3.1 -/- ; 3.2 -/- double knock-out mice. In contrast, smooth-muscle specific deletion of the L-type VGCC, Ca v 1.2, completely abolished all lymphatic spontaneous contractions. Collectively our results suggest that, although T-type VGCCs are expressed in mouse lymphatic smooth muscle, they do not play a significant role in modulating the frequency of the ionic pacemaker or the amplitude of spontaneous contractions. We conclude that the effects of mibefradil and Ni 2+ in other lymphatic preparations are largely or completely explained by off-target effects on L-type VGCCs, which are essential for controlling both the frequency and strength of spontaneous contractions.

T-type Ca 2+ channel Ca v 3.1 promotes microvessel contraction ex vivo. It was hypothesized that in vivo, functional deletion of Ca v 3.1, but not Ca v 3.2, protects mice against angiotensin II (ANG II)-induced hypertension. Mean arterial blood pressure (MAP) and heart rate were measured continuously with chronically indwelling catheters during infusion of ANG II (30 ng·kg -1 ·min -1 , 7 days) in wild-type (WT), Ca v 3.1 -/- , and Ca v 3.2 -/- mice. Plasma aldosterone and renin concentrations were measured by radioimmunoassays. In a separate series, WT mice were infused with ANG II (100 ng·kg -1 ·min -1 ) with and without the mineralocorticoid receptor blocker canrenoate. Ca v 3.1 -/- and Ca v 3.2 -/- mice exhibited no baseline difference in MAP compared with WT mice, but day-night variation was blunted in both Ca v 3.1 and Ca v 3.2 -/- mice. ANG II increased significantly MAP in WT, Ca v 3.1 -/- , and Ca v 3.2 -/- mice with no differences between genotypes. Heart rate was significantly lower in Ca v 3.1 -/- and Ca v 3.2 -/- mice compared with control mice. After ANG II infusion, plasma aldosterone concentration was significantly lower in Ca v 3.1 -/- compared with Ca v 3.2 -/- mice. In response to ANG II, fibrosis was observed in heart sections from both WT and Ca v 3.1 -/- mice and while cardiac atrial natriuretic peptide mRNA was similar, the brain natriuretic peptide mRNA increase was mitigated in Ca v 3.1 -/- mice ANG II at 100 ng/kg yielded elevated pressure and an increased heart weight-to-body weight ratio in WT mice. Cardiac hypertrophy, but not hypertension, was prevented by the mineralocorticoid receptor blocker canrenoate. In conclusion, T-type channels Ca v 3.1and Ca v 3.2 do not contribute to baseline blood pressure levels and ANG II-induced hypertension. Ca v 3.1, but not Ca v 3.2, contributes to aldosterone secretion. Aldosterone promotes cardiac hypertrophy during hypertension.

Objectives: Voltage-gated Ca 2+ channels (VGCCs) are of central relevance in regulating Ca 2+ influx into living cells. The low-voltage activated (LVA) Ca v 3 T-type Ca 2+ channels are widely distributed throughout the brain including the peripheral auditory system and ascending auditory tract. Their exact role in auditory information processing is still not fully understood. Within the LVA subgroup, Ca v 3.2 T-type Ca 2+ channels seem to be of special importance as qPCR revealed a steady increase in Ca v 3.2 transcript levels over age, e.g. in the cochlea and spiral ganglion neurons (SGN). Furthermore, pharmacological studies suggested an association between Ca v 3.2 expression and both age-related and noise-induced hearing loss. Given the potential functional relevance of Ca v 3.2 VGGCs in sensorineural hearing loss, we recorded gender specific auditory evoked brainstem responses (ABRs) upon both click and tone burst presentation. Here we present auditory brainstem response (ABR) data from Ca v 3.2 +/+ , Ca v 3.2 +/- and Ca v 3.2 -/- mice from both genders which are of value for researchers who want to evaluate how Ca v 3.2 loss affects basic auditory parameters, e.g. click and tone burst based hearing thresholds, amplitude growth function and peak latencies., Data Description: Information presented here includes ABR data from age-matched female and male Ca v 3.2 +/+ , Ca v 3.2 +/- and Ca v 3.2 -/- mice and technical aspects of the auditory recording protocol. Data were recorded using a commercially available ABR setup from Tucker Davis Technologies Inc. (TDT). Raw data files (arf.-file format) were exported as txt.-files with free access for analysis.

Bortezomib, a first-line agent for treatment of multiple myeloma, exhibits anticancer activity through proteasome inhibition. However, bortezomib-induced peripheral neuropathy (BIPN) is one of the most serious side effects. Since decreased proteasomal degradation of Ca v 3.2 T-type calcium channels in the primary afferents is involved in persistent pain, we investigated whether BIPN involves increased protein levels of Ca v 3.2 in mice. Six repeated i.p. administrations of bortezomib for 12 days developed persistent mechanical allodynia. Systemic administration of novel T-type calcium channel blockers, (2R/S)-6-prenylnaringenin and KTt-45, and of TTA-A2, the well-known blocker, reversed the BIPN. Ascorbic acid, known to block Ca v 3.2, but not Ca v 3.1 or 3.3, and silencing of Ca v 3.2 gene also suppressed BIPN. Protein levels of Ca v 3.2 in the dorsal root ganglion (DRG) at L4-L6 levels increased throughout days 1-21 after the onset of bortezomib treatment. Protein levels of USP5, a deubiquitinating enzyme that specifically inhibits proteasomal degradation of Ca v 3.2, increased in DRG on days 3-21, but not day 1, in bortezomib-treated mice. In DRG-derived ND7/23 cells, bortezomib increased protein levels of Ca v 3.2 and T-channel-dependent currents, as assessed by a patch-clamp method, but did not upregulate expression of Ca v 3.2 mRNA or USP5 protein. MG-132, another proteasome inhibitor, also increased Ca v 3.2 protein levels in the cultured cells. Given the previous evidence for USP5 induction following nociceptor excitation, our data suggest that BIPN involves the increased protein levels of Ca v 3.2 in nociceptors through inhibition of proteasomal degradation of Ca v 3.2 by bortezomib itself and then by USP5 that is upregulated probably in an activity-dependent manner., (Copyright © 2018 Elsevier B.V. All rights reserved.)

T-type calcium channels in the brain mediate the pathophysiology of epilepsy, pain, and sleep. Recently, we developed a novel therapeutic candidate, SAK3 (ethyl 8'-methyl-2',4-dioxo-2-(piperidin-1-yl)-2'H-spiro[cyclopentane-1,3'-imidazo[1,2-a] pyridine]-2-ene-3-carboxylate), for Alzheimer's disease (AD). The cognitive improvement by SAK3 is closely associated with enhanced acetylcholine (ACh) release in the hippocampus. Since monoamines such as dopamine (DA), noradrenaline (NA), and serotonin (5-HT) are also involved in hippocampus-dependent learning and psychomotor behaviors in mice, we investigated the effects of SAK3 on these monoamine releases in the mouse brain. Oral administration of SAK3 (0.5 mg/kg, p.o.) significantly promoted DA and 5-HT releases in the naive mouse hippocampal CA1 region but not in the medial prefrontal cortex (mPFC), while SAK3 did not affect NA release in either brain region. The T-type calcium channel-specific inhibitor, NNC 55-0396 (1 μM) significantly antagonized SAK3-enhanced DA and 5-HT releases in the hippocampus. Interestingly, the α7 nicotinic ACh receptor (nAChR) antagonist, methyllycaconitine (1 nM) significantly inhibited DA release, and the α4 nAChR antagonist, dihydro-β-erythroidine (100 μM) significantly blocked both DA and 5-HT releases following SAK3 (0.5 mg/kg, p.o.) administration in the hippocampus. SAK3 did not alter basal monoamine contents both in the mPFC and hippocampus. SAK3 (0.5 mg/kg, p.o.) administration also significantly elevated DA and 5-HT releases in the hippocampal CA1 region of amyloid-precursor protein (APP)NL-GF knock-in (KI) mice. Moreover, hippocampal DA and 5-HT contents were significantly decreased in APPNL-GF KI mice. Taken together, our data suggest that SAK3 promotes monoamine DA and 5-HT releases by enhancing the T-type calcium channel and nAChR in the mouse hippocampus., Competing Interests: The authors have declared that no competing interests exist.

Objective- This study examined whether caveolae position Ca V 3.2 (T-type Ca2+ channel encoded by the α-3.2 subunit) sufficiently close to RyR (ryanodine receptors) for extracellular Ca 2+ influx to trigger Ca 2+ sparks and large-conductance Ca 2+ -activated K + channel feedback. Approach and Results- Using smooth muscle cells from mouse mesenteric arteries, the proximity ligation assay confirmed that Ca V 3.2 reside within 40 nm of caveolin 1, a key caveolae protein. Methyl-β-cyclodextrin, a cholesterol depleting agent that disrupts caveolae, suppressed Ca V 3.2 activity along with large-conductance Ca 2+ -activated K + -mediated spontaneous transient outward currents in cells from C57BL/6 but not Ca V 3.2 -/- mice. Genetic deletion of caveolin 1, a perturbation that prevents caveolae formation, also impaired spontaneous transient outward current production but did so without impairing Ca 2+ channel activity, including Ca V 3.2. These observations indicate a mistargeting of Ca V 3.2 in caveolin 1 -/- mice, a view supported by a loss of Ni 2+ -sensitive Ca 2+ spark generation and colocalization signal (Ca V 3.2-RyR) from the proximity ligation assay. Vasomotor and membrane potential measurements confirmed that cellular disruption of the Ca V 3.2-RyR axis functionally impaired the ability of large-conductance Ca 2+ -activated K + to set tone in pressurized caveolin 1 -/- arteries. Conclusions- Caveolae play a critical role in protein targeting and preserving the close structural relationship between Ca V 3.2 and RyR needed to drive negative feedback control in resistance arteries.

Mature granule cells are poorly excitable neurons that were recently shown to fire action potentials, preferentially in bursts. It is believed that the particularly pronounced short-term facilitation of mossy fiber synapses makes granule cell bursting a very effective means of properly transferring information to CA3. However, the mechanism underlying the unique bursting behavior of mature granule cells is currently unknown. Here, we show that Cav3.2 T-type channels at the axon initial segment are responsible for burst firing of mature granule cells in rats and mice. Accordingly, Cav3.2 knockout mice fire tonic spikes and exhibit impaired bursting, synaptic plasticity and dentate-to-CA3 communication. The data show that Cav3.2 channels are strong modulators of bursting and can be considered a critical molecular switch that enables effective information transfer from mature granule cells to the CA3 pyramids.

Aims: To obtain functional evidence that ICa,T is involved in the pathogenesis of cardiac hypertrophy and heart failure. We unexpectedly identified ICa(TTX) rather than ICa,T, therefore, we adjusted our aim to encompass these findings., Methods and Results: We investigated (1) Cav3.1 (α1G) transgenic (Tg) mice compared with nontransgenic (tTA-Ntg); (2) Cav3.1-deficient mice (Cav3.1) compared with wild type (Wt) after chemically and surgically induced cardiac remodeling; and (3) spontaneous hypertensive rats and thoracic aortic constriction (TAC) rats. Whole-cell patch-clamp technique was used to measure ICa in ventricular myocytes. Cav3.1-Tg expressed ICa,T (-18.35 ± 1.02 pA/pF at -40 mV) without signs of compromised cardiac function. While we failed to detect ICa,T after hypertrophic stimuli, instead we demonstrated that both Wt and Cav3.1 mouse exhibit ICa(TTX). Using TAC rats, only 2 of 24 VMs showed ICa,T under our experimental conditions. Without TTX, ICa(TTX) occurred in VMs from Wt, spontaneous hypertensive rats, and TAC rats also., Conclusions: These findings demonstrate for the first time that mouse VMs express ICa(TTX). We suggest that future studies should take into consideration the measuring conditions when interpreting ICa,T reappearance in ventricular myocytes in response to hypertrophic stress. Contamination with ICa(TTX) could possibly confuse the relevance of the data.

Exploratory drive is one of the most fundamental emotions, of all organisms, that are evoked by novelty stimulation. Exploratory behavior plays a fundamental role in motivation, learning, and well-being of organisms. Diverse exploratory behaviors have been described, although their heterogeneity is not certain because of the lack of solid experimental evidence for their distinction. Here we present results demonstrating that different neural mechanisms underlie different exploratory behaviors. Localized Cav3.1 knockdown in the medial septum (MS) selectively enhanced object exploration, whereas the null mutant (KO) mice showed enhanced-object exploration as well as open-field exploration. In MS knockdown mice, only type 2 hippocampal theta rhythm was enhanced, whereas both type 1 and type 2 theta rhythm were enhanced in KO mice. This selective effect was accompanied by markedly increased excitability of septo-hippocampal GABAergic projection neurons in the MS lacking T-type Ca(2+) channels. Furthermore, optogenetic activation of the septo-hippocampal GABAergic pathway in WT mice also selectively enhanced object exploration behavior and type 2 theta rhythm, whereas inhibition of the same pathway decreased the behavior and the rhythm. These findings define object exploration distinguished from open-field exploration and reveal a critical role of T-type Ca(2+) channels in the medial septal GABAergic projection neurons in this behavior.

Study Objectives: Low-threshold voltage-gated T-type Ca(2+) channels (T-channels or CaV3 channels) sustain oscillatory discharges of thalamocortical (TC) and nucleus Reticularis thalami (nRt) cells. The CaV3.3 subtype dominates nRt rhythmic bursting and mediates a substantial fraction of spindle power in the NREM sleep EEG. CaV3.2 channels are also found in nRt, but whether these contribute to nRt-dependent spindle generation is unexplored. We investigated thalamic rhythmogenesis in mice lacking this subtype in isolation (CaV3.2KO mice) or in concomitance with CaV3.3 deletion (CaV3.double-knockout (DKO) mice)., Methods: We examined discharge characteristics of thalamic cells and intrathalamic evoked synaptic transmission in brain slices from wild-type, CaV3.2KO and CaV3.DKO mice through patch-clamp recordings. The sleep profile of freely behaving CaV3.2KO and CaV3.DKO mice was assessed by polysomnographic recordings., Results: CaV3.2 channel deficiency left nRt discharge properties largely unaltered, but additional deletion of CaV3.3 channels fully abolished low-threshold whole-cell Ca(2+) currents and bursting, and suppressed burst-mediated inhibitory responses in TC cells. CaV3.DKO mice had more fragmented sleep, with shorter NREM sleep episodes and more frequent microarousals. The NREM sleep EEG power spectrum displayed a relative suppression of the σ frequency band (10-15 Hz), which was accompanied by an increase in the δ band (1-4 Hz)., Conclusions: Consistent with previous findings, CaV3.3 channels dominate nRt rhythmogenesis, but the lack of CaV3.2 channels further aggravates neuronal, synaptic, and EEG deficits. Therefore, CaV3.2 channels can boost intrathalamic synaptic transmission, and might play a modulatory role adjusting the relative presence of NREM sleep EEG rhythms., (© 2016 Associated Professional Sleep Societies, LLC.)

A crucial pathophysiological issue concerning central neuropathic pain is the modification of sensory processing by abnormally increased low-frequency brain rhythms. Here we explore the molecular mechanisms responsible for such abnormal rhythmicity and its relation to neuropathic pain syndrome. Toward this aim, we investigated the behavioral and electrophysiological consequences of trigeminal neuropathic pain following infraorbital nerve ligations in CaV3.1 T-type Ca(2+) channel knockout and wild-type mice. CaV3.1 knockout mice had decreased mechanical hypersensitivity and reduced low-frequency rhythms in the primary somatosensory cortex and related thalamic nuclei than wild-type mice. Lateral inhibition of gamma rhythm in primary somatosensory cortex layer 4, reflecting intact sensory contrast, was present in knockout mice but severely impaired in wild-type mice. Moreover, cross-frequency coupling between low-frequency and gamma rhythms, which may serve in sensory processing, was pronounced in wild-type mice but not in CaV3.1 knockout mice. Our results suggest that the presence of CaV3.1 channels is a key element in the pathophysiology of trigeminal neuropathic pain.

Objective: In resistance arteries, there is an emerging view that smooth muscle CaV3.2 channels restrain arterial constriction through a feedback response involving the large-conductance Ca(2+)-activated K(+) channel (BKCa). Here, we used wild-type and CaV3.2 knockout (CaV3.2(-/-)) mice to definitively test whether CaV3.2 moderates myogenic tone in mesenteric arteries via the CaV3.2-ryanodine receptor-BKCa axis and whether this regulatory mechanism influences blood pressure regulation., Approach and Results: Using pressurized vessel myography, CaV3.2(-/-) mesenteric arteries displayed enhanced myogenic constriction to pressure but similar K(+)-induced vasoconstriction compared with wild-type C57BL/6 arteries. Electrophysiological and myography experiments subsequently confirmed the inability of micromolar Ni(2+), a CaV3.2 blocker, to either constrict arteries or suppress T-type currents in CaV3.2(-/-) smooth muscle cells. The frequency of BKCa-induced spontaneous transient outward K(+) currents dropped in wild-type but not in knockout arterial smooth muscle cells upon the pharmacological suppression of CaV3.2 channel. Line scan analysis performed on en face arteries loaded with Fluo-4 revealed the presence of Ca(2+) sparks in all arteries, with the subsequent application of Ni(2+) only affecting wild-type arteries. Although CaV3.2 channel moderated myogenic constriction of resistance arteries, the blood pressure measurements of CaV3.2(-/-) and wild-type animals were similar., Conclusions: Overall, our findings establish a negative feedback mechanism of the myogenic response in which CaV3.2 channel modulates downstream ryanodine receptor-BKCa to hyperpolarize and relax arteries., (© 2015 American Heart Association, Inc.)

Atypical absence epilepsy (AAE) is an intractable disorder characterized by slow spike-and-wave discharges in electroencephalograms (EEGs) and accompanied by severe cognitive dysfunction and neurodevelopmental or neurological deficits in humans. Administration of the cholesterol biosynthesis inhibitor AY-9944 (AY) during the postnatal developmental period induces AAE in animals; however, the neural mechanism of seizure development remains largely unknown. In this study, we characterized the cellular manifestations of AY-induced AAE in the mouse. Treatment of brain slices with AY increased membrane excitability of hippocampal CA1 neurons. AY treatment also increased input resistance of CA1 neurons during early postnatal days (PND) 5-10. However, these effects were not observed during late PND (14-21) or in adulthood (7-10 weeks). Notably, AY treatment elicited paroxysmal depolarizing shift (PDS)-like epileptiform discharges during the early postnatal period, but not during late PND or in adults. The PDS-like events were not compromised by application of glutamate or GABA receptor antagonists. However, the PDS-like events were abolished by blockage of voltage-gated Na(+) channels. Hippocampal neurons isolated from an in vivo AY model of AAE showed similar PDS-like epileptiform discharges. Further, AY-treated neurons from T-type Ca(2+) channel α1G knockout (Cav3.1(-/-)) mice, which do not exhibit typical absence seizures, showed similar PDS-like epileptiform discharges. These results demonstrate that PDS-like epileptiform discharges during the early postnatal period are dependent upon Na(+) channels and are involved in the generation of AY-induced AAE, which is distinct from typical absence epilepsy. Our findings may aid our understanding of the pathophysiological mechanisms of clinical AAE in individuals, such as those with Lennox-Gastaut syndrome., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Homozygous Cav3.2 knockout mice, which are defective in the pore-forming subunit of a low voltage activated T-type calcium channel, have been documented to show impaired maintenance of late-phase long-term potentiation (L-LTP) and defective retrieval of context-associated fear memory. To investigate the role of Cav3.2 in global gene expression, we performed a microarray transcriptome study on the hippocampi of the Cav3.2-/- mice and their wild-type littermates, either naïve (untrained) or trace fear conditioned. We found a significant left-right asymmetric effect on the hippocampal transcriptome caused by the Cav3.2 knockout. Between the naive Cav3.2-/- and the naive wild-type mice, 3522 differentially expressed genes (DEGs) were found in the left hippocampus, but only 4 DEGs were found in the right hippocampus. Remarkably, the effect of Cav3.2 knockout was partially reversed by trace fear conditioning. The number of DEGs in the left hippocampus was reduced to 6 in the Cav3.2 knockout mice after trace fear conditioning, compared with the wild-type naïve mice. To our knowledge, these results demonstrate for the first time the asymmetric effects of the Cav3.2 and its partial reversal by behavior training on the hippocampal transcriptome.

Early in development, before the onset of vision, the retina establishes direction-selective responses. During this time period, the retina spontaneously generates bursts of action potentials that propagate across its extent. The precise spatial and temporal properties of these "retinal waves" have been implicated in the formation of retinal projections to the brain. However, their role in the development of direction selective circuits within the retina has not yet been determined. We addressed this issue by combining multielectrode array and cell-attached recordings to examine mice that lack the CaV3.2 subunit of T-type Ca2+ channels (CaV3.2 KO) because these mice exhibit disrupted waves during the period that direction selective circuits are established. We found that the spontaneous activity of these mice displays wave-associated bursts of action potentials that are altered from that of control mice: the frequency of these bursts is significantly decreased and the firing rate within each burst is reduced. Moreover, the projection patterns of the retina demonstrate decreased eye-specific segregation in the dorsal lateral geniculate nucleus (dLGN). However, after eye-opening, the direction selective responses of CaV3.2 KO direction selective ganglion cells (DSGCs) are indistinguishable from those of wild-type DSGCs. Our data indicate that although the temporal properties of the action potential bursts associated with retinal waves are important for activity-dependent refining of retinal projections to central targets, they are not critical for establishing direction selectivity in the retina.

Intrinsic burst and rhythmic burst discharges (RBDs) are elicited by activation of T-type Ca(2+) channels in the thalamic reticular nucleus (TRN). TRN bursts are believed to be critical for generation and maintenance of thalamocortical oscillations, leading to the spike-and-wave discharges (SWDs), which are the hallmarks of absence seizures. We observed that the RBDs were completely abolished, whereas tonic firing was significantly increased, in TRN neurons from mice in which the gene for the T-type Ca(2+) channel, CaV3.3, was deleted (CaV3.3(-/-)). Contrary to expectations, there was an increased susceptibility to drug-induced SWDs both in CaV3.3(-/-) mice and in mice in which the CaV3.3 gene was silenced predominantly in the TRN. CaV3.3(-/-) mice also showed enhanced inhibitory synaptic drive onto TC neurons. Finally, a double knockout of both CaV3.3 and CaV3.2, which showed complete elimination of burst firing and RBDs in TRN neurons, also displayed enhanced drug-induced SWDs and absence seizures. On the other hand, tonic firing in the TRN was increased in these mice, suggesting that increased tonic firing in the TRN may be sufficient for drug-induced SWD generation in the absence of burst firing. These results call into question the role of burst firing in TRN neurons in the genesis of SWDs, calling for a rethinking of the mechanism for absence seizure induction.

To exert its analgesic action, paracetamol requires complex metabolism to produce a brain-specific lipoamino acid compound, AM404, which targets central transient receptor potential vanilloid receptors (TRPV1). Lipoamino acids are also known to induce analgesia through T-type calcium-channel inhibition (Ca(v)3.2). In this study we show that the antinociceptive effect of paracetamol in mice is lost when supraspinal Ca(v)3.2 channels are inhibited. Therefore, we hypothesized a relationship between supraspinal Ca(v)3.2 and TRPV1, via AM404, which mediates the analgesic effect of paracetamol. AM404 is able to activate TRPV1 and weakly inhibits Ca(v)3.2. Interestingly, activation of TRPV1 induces a strong inhibition of Ca(v)3.2 current. Supporting this, intracerebroventricular administration of AM404 or capsaicin produces antinociception that is lost in Ca(v)3.2(-/-) mice. Our study, for the first time, (1) provides a molecular mechanism for the supraspinal antinociceptive effect of paracetamol; (2) identifies the relationship between TRPV1 and the Ca(v)3.2 channel; and (3) suggests supraspinal Ca(v)3.2 inhibition as a potential pharmacological strategy to alleviate pain., (Copyright © 2014 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Interstitial cells of Cajal (ICC) generate slow waves in gastrointestinal (GI) muscles. Previous studies have suggested that slow wave generation and propagation depends on a voltage-dependent Ca(2+) entry mechanism with the signature of a T-type Ca(2+) conductance. We studied voltage-dependent inward currents in isolated ICC. ICC displayed two phases of inward current upon depolarization: a low voltage-activated inward current and a high voltage-activated current. The latter was of smaller current density and blocked by nicardipine. Ni(2+) (30 μM) or mibefradil (1 μM) blocked the low voltage-activated current. Replacement of extracellular Ca(2+) with Ba(2+) did not affect the current, suggesting that either charge carrier was equally permeable. Half-activation and half-inactivation occurred at -36 and -59 mV, respectively. Temperature sensitivity of the Ca(2+) current was also characterized. Increasing temperature (20-30°C) augmented peak current from -7 to -19 pA and decreased the activation time from 20.6 to 7.5 ms [temperature coefficient (Q10) = 3.0]. Molecular studies showed expression of Cacna1g (Cav3.1) and Cacna1h (Cav3.2) in ICC. The temperature dependence of slow waves in intact jejunal muscles of wild-type and Cacna1h(-/-) mice was tested. Reducing temperature decreased the upstroke velocity significantly. Upstroke velocity was also reduced in muscles of Cacna1h(-/-) mice, and Ni(2+) or reduced temperature had little effect on these muscles. Our data show that a T-type conductance is expressed and functional in ICC. With previous studies our data suggest that T-type current is required for entrainment of pacemaker activity within ICC and for active propagation of slow waves in ICC networks.

Unlabelled: Epithelial tumor cells express T-type Ca(2+) channels, which are thought to promote cell proliferation. This study investigated the cellular response to T-type Ca(2+) channel inhibition either by small-molecule antagonists or by RNAi-mediated knockdown. Selective T-type Ca(2+) channel antagonists caused growth inhibition and apoptosis more effectively in HCT116 cells expressing wild-type p53 (p53wt), than in HCT116 mutant p53(-/-) cells. These antagonists increased p53-dependent gene expression and increased genomic occupancy of p53 at specific target sequences. The knockdown of a single T-type Ca(2+) channel subunit (CACNA1G) reduced cell growth and induced caspase-3/7 activation in HCT116 p53wt cells as compared with HCT116 mutant p53(-/-) cells. Moreover, CaCo2 cells that do not express functional p53 were made more sensitive to CACNA1G knockdown when p53wt was stably expressed. Upon T-type Ca(2+) channel inhibition, p38-MAPK promoted phosphorylation at Ser392 of p53wt. Cells treated with the inhibitor SB203580 or specific RNAi targeting p38-MAPKα/β (MAPK14/MAPK11) showed resistance to T-type Ca(2+) channel inhibition. Finally, the decreased sensitivity to channel inhibition was associated with decreased accumulation of p53 and decreased expression of p53 target genes, p21Cip1 (CDKN1A) and BCL2-binding component 3 (BBC3/PUMA)., Implications: A novel pathway involving p53 and p38-MAPK is revealed and provides a rationale for antitumor therapies that target T-type Ca(2+) channels.

Background: When complete atrioventricular block (AVB) occurs, infranodal escape rhythms are essential to prevent bradycardic death. The role of T-type Ca(2+) channels in pacemaking outside the sinus node is unknown. We investigated the role of T-type Ca(2+) channels in escape rhythms and bradycardia-related ventricular tachyarrhythmias after AVB in mice., Methods and Results: Adult male mice lacking the main T-type Ca(2+) channel subunit Cav3.1 (Cav3.1(-/-)) and wild-type (WT) controls implanted with ECG telemetry devices underwent radiofrequency atrioventricular node ablation to produce AVB. Before ablation, Cav3.1(-/-) mice showed sinus bradycardia (mean±SEM; RR intervals, 148±3 versus 128±2 ms WT; P<0.001). Immediately after AVB, Cav3.1(-/-) mice had slower escape rhythms (RR intervals, 650±75 versus 402±26 ms in WT; P<0.01) but a preserved heart-rate response to isoproterenol. Over the next 24 hours, mortality was markedly greater in Cav3.1(-/-) mice (19/31; 61%) versus WT (8/26; 31%; P<0.05), and Torsades de Pointes occurred more frequently (73% Cav3.1(-/-) versus 35% WT; P<0.05). Escape rhythms improved in both groups during the next 4 weeks but remained significantly slower in Cav3.1(-/-). At 4 weeks after AVB, ventricular tachycardia was more frequent in Cav3.1(-/-) than in WT mice (746±116 versus 214±78 episodes/24 hours; P<0.01). Ventricular function remodeling was similar in Cav3.1(-/-) and WT, except for smaller post-AVB fractional-shortening increase in Cav3.1(-/-). Expression changes were seen post-AVB for a variety of genes; these tended to be greater in Cav3.1(-/-) mice, and overexpression of fetal and profibrotic genes occurred only in Cav3.1(-/-)., Conclusions: This study suggests that T-type Ca(2+) channels play an important role in infranodal escape automaticity. Loss of T-type Ca(2+) channels worsens bradycardia-related mortality, increases bradycardia-associated adverse remodeling, and enhances the risk of malignant ventricular tachyarrhythmias complicating AVB.

Aims: As cardiovascular disease is characterized by reduced nitric oxide bioavailability, our aim was to determine the impact of this change on the mechanism underlying vascular tone of pressurized arteries in vitro and in vivo., Methods and Results: We used pressurized cerebral and mesenteric arteries in vitro and skeletal muscle arterioles in vivo to study the contribution of L-type (1 µmol/L nifedipine) and T-type (1 µmol/L mibefradil, 3 µmol/L NNC 55-0396) calcium channels to vascular tone, following acute or chronic inhibition of nitric oxide. Acute inhibition with l-NAME (10 µmol/L) significantly increased the T-type, but not the L-type, channel contribution to vascular tone in vitro and in vivo, and altered the smooth muscle expression of the Cav3.1 and Cav3.2 T-type channels. In pressurized mesenteric arteries of Cav3.1ko and Cav3.2ko mice, acutely treated with l-NAME, the contribution of T-type channels relative to L-type channels was significantly reduced, compared with arteries from wild-type mice.Chronic l-NAME treatment (40 mg/kg/day; 14-18 days) increased blood pressure, vascular superoxide, and the contribution of T-type channels to vascular tone in vivo. The latter was reversed by acute scavenging of superoxide with tempol (1 mmol/L), or inhibition of NADPH oxidase with apocynin (500 µmol/L) or DPI (5 µmol/L)., Conclusion: We conclude that nitric oxide deficit produces a significant increase in the contribution of Cav3.1 and Cav3.2 T-type calcium channels to vascular tone, by regulating the bioavailability of reactive oxygen species produced by NADPH oxidase. Our data provide evidence for a novel causal link between nitric oxide deficit, oxidative stress, and T-type calcium channel function.

Aim: Using mice deficient in the CaV 3.1 T-type Ca(2+) channel, the aim of the present study was to elucidate the molecular identity of non-L-type channels involved in vascular tone regulation in mesenteric arteries and arterioles., Methods: We used immunofluorescence microscopy to localize CaV 3.1 channels, patch clamp electrophysiology to test the effects of a putative T-type channel blocker NNC 55-0396 on whole-cell Ca(2+) currents, pressure myography and Ca(2+) imaging to test diameter and Ca(2+) responses of the applied vasoconstrictors, and Q-PCR to check mRNA expression levels of several Ca(2+) handling proteins in wild-type and CaV 3.1(-/-) mice., Results: Our data indicated that CaV 3.1 channels are important for the maintenance of myogenic tone at low pressures (40-80 mm Hg), whereas they are not involved in high-voltage-activated Ca(2+) currents, Ca(2+) entry or vasoconstriction to high KCl in mesenteric arteries and arterioles. Furthermore, we show that NNC 55-0396 is not a specific T-type channel inhibitor, as it potently blocks L-type and non-L-type high-voltage-activated Ca(2+) currents in mouse mesenteric vascular smooth muscle cell., Conclusion: Our data using mice deficient in the CaV 3.1 T-type channel represent new evidence for the involvement of non-L-type channels in arteriolar tone regulation. We showed that CaV 3.1 channels are important for the myogenic tone at low arterial pressure, which is potentially relevant under resting conditions in vivo. Moreover, CaV 3.1 channels are not involved in Ca(2+) entry and vasoconstriction to large depolarization with, for example, high KCl. Finally, we caution against using NNC 55-0396 as a specific T-type channel blocker in native cells expressing high-voltage-activated Ca(2+) channels., (Acta Physiologica © 2013 Scandinavian Physiological Society.)

Aims: Restenosis is an undesirable consequence following percutaneous vascular interventions. However, the current strategy for preventing restenosis is inadequate. The aim of this study was to investigate the role of low-voltage gated T-type calcium channels in regulating vascular smooth muscle cell (VSMC) proliferation during neointimal formation., Methods and Results: Wire injury of mice carotid arteries resulted in neointimal formation in the wild-type and Ca(v)3.2(-/-) but not Ca(v)3.1(-/-) mice, indicating a critical role of Ca(v)3.1 in neointimal formation. In addition, we found a significant increase of Ca(v)3.1 mRNA and protein in injured arteries. Ca(v)3.1 knockout or knockdown (shCa(v)3.1) reduced VSMC proliferation. Since T-channels are expressed predominantly in the G(1) and S phases in VSMCs, we examined whether an abnormal G(1)/S transition was the cause of the reduced cell proliferation in shCa(v)3.1 VSMCs. We found a disrupted expression of cyclin E in shCa(v)3.1 VSMCs, and calmodulin agonist CALP1 partially rescued the defective cell proliferation. Furthermore, we demonstrated that infusion of NNC55-0396, a selective T-channel blocker, inhibited neointimal formation in wild-type mice., Conclusion: Ca(v)3.1 is required for VSMC proliferation during neointimal formation, and blocking of Ca(v)3.1 may be beneficial for preventing restenosis.

Among all voltage-gated calcium channels, the T-type Ca²⁺ channels encoded by the Ca(v)3.2 genes are highly expressed in the hippocampus, which is associated with contextual, temporal and spatial learning and memory. However, the specific involvement of the Ca(v)3.2 T-type Ca²⁺ channel in these hippocampus-dependent types of learning and memory remains unclear. To investigate the functional role of this channel in learning and memory, we subjected Ca(v)3.2 homozygous and heterozygous knockout mice and their wild-type littermates to hippocampus-dependent behavioral tasks, including trace fear conditioning, the Morris water-maze and passive avoidance. The Ca(v)3.2 ⁻/⁻ mice performed normally in the Morris water-maze and auditory trace fear conditioning tasks but were impaired in the context-cued trace fear conditioning, step-down and step-through passive avoidance tasks. Furthermore, long-term potentiation (LTP) could be induced for 180 minutes in hippocampal slices of WTs and Ca(v)3.2 ⁺/⁻ mice, whereas LTP persisted for only 120 minutes in Ca(v)3.2 ⁻/⁻ mice. To determine whether the hippocampal formation is responsible for the impaired behavioral phenotypes, we next performed experiments to knock down local function of the Ca(v)3.2 T-type Ca²⁺ channel in the hippocampus. Wild-type mice infused with mibefradil, a T-type channel blocker, exhibited similar behaviors as homozygous knockouts. Taken together, our results demonstrate that retrieval of context-associated memory is dependent on the Ca(v)3.2 T-type Ca²⁺ channel.

Aims: Previous studies suggested that T-type Ca(2+)-current (I(CaT))-blockers improve cardiac remodelling, but all available I(CaT)-blockers have non-specific actions on other currents and/or functions. To clarify the role of I(CaT) in cardiac remodelling, we studied mice with either of the principal cardiac I(CaT)-subunits (Cav3.1 or Cav3.2) knocked out., Methods and Results: Adult male Cav3.1- or Cav3.2-knockout (Cav3.1(-/-), Cav3.2(-/-)) mice and respective wild-type (WT) littermate controls were subjected to left anterior descending coronary artery ligation to create myocardial infarction (MI). Echocardiography and programmed electrical stimulation were performed at baseline and 4 weeks post-MI. At baseline, Cav3.1(-/-) mice had slowed heart rates and longer PR intervals vs. WT, but no other electrophysiological and no haemodynamic differences. Cav3.2(-/-) showed no differences vs. WT. Contractile indices (left ventricular fractional shortening and ejection fraction) decreased more post-MI in Cav3.1(-/-) mice than in Cav3.1(+/+) (e.g. by 34 and 29% for WT; 50 and 45% for Cav3.1(-/-), respectively; P < 0.05 for each). Cav3.1(-/-) mice had increased ventricular tachycardia (VT) inducibility post-MI (9 of 11, 82%) vs. WT (3 of 10, 30%; P < 0.05). Cav3.2(-/-) mice were not different in cardiac function or VT inducibility vs. WT. Quantitative polymerase chain reaction showed that Cav3.1 is the major I(CaT)-subunit and that no compensatory Cav3.2 up-regulation occurs in Cav3.1(-/-) mice. Cav3.1(-/-) and Cav3.2(-/-) mice had no mRNA expression for the knocked-out gene, at baseline or post-MI., Conclusion: Our findings suggest that, contrary to suggestions from previous studies with (imperfectly selective) pharmacological agents having T-type Ca(2+)-channel-blocking actions, elimination of Cav3.1 expression leads to impaired cardiac function and enhanced arrhythmia vulnerability post-MI, whereas Cav3.2 elimination has no effect.

Loss of spiral ganglion neurons is a major cause of age-related hearing loss (presbycusis). Despite being the third most prevalent condition afflicting elderly persons, there are no known medications to prevent presbycusis. Because calcium signaling has long been implicated in age-related neuronal death, we investigated T-type calcium channels. This family is comprised of three members (Ca(v)3.1, Ca(v)3.2, and Ca(v)3.3), based on their respective main pore-forming alpha subunits: α1G, α1H, and α1I. In the present study, we report a significant delay of age-related loss of cochlear function and preservation of spiral ganglion neurons in α1H null and heterozygous mice, clearly demonstrating an important role for Ca(v)3.2 in age-related neuronal loss. Furthermore, we show that anticonvulsant drugs from a family of T-type calcium channel blockers can significantly preserve spiral ganglion neurons during aging. To our knowledge, this is the first report of drugs capable of diminishing age-related loss of spiral ganglion neurons., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The symptoms of irritable bowel syndrome (IBS) include significant abdominal pain and bloating. Current treatments are empirical and often poorly efficacious, and there is a need for the development of new and efficient analgesics aimed at IBS patients. T-type calcium channels have previously been validated as a potential target to treat certain neuropathic pain pathologies. Here we report that T-type calcium channels encoded by the Ca(V)3.2 isoform are expressed in colonic nociceptive primary afferent neurons and that they contribute to the exaggerated pain perception in a butyrate-mediated rodent model of IBS. Both the selective genetic inhibition of Ca(V)3.2 channels and pharmacological blockade with calcium channel antagonists attenuates IBS-like painful symptoms. Mechanistically, butyrate acts to promote the increased insertion of Ca(V)3.2 channels into primary sensory neuron membranes, likely via a posttranslational effect. The butyrate-mediated regulation can be recapitulated with recombinant Ca(V)3.2 channels expressed in HEK cells and may provide a convenient in vitro screening system for the identification of T-type channel blockers relevant to visceral pain. These results implicate T-type calcium channels in the pathophysiology of chronic visceral pain and suggest Ca(V)3.2 as a promising target for the development of efficient analgesics for the visceral discomfort and pain associated with IBS.

Background: Mice that have defects in their low-threshold T-type calcium channel (T-channel) genes show altered pain behaviors. The changes in the ratio of nociceptive neurons and the burst firing property of reticular thalamic (RT) and ventroposterior (VP) neurons in Cav3.2 knockout (KO) mice were studied to test the involvement of thalamic T-channel and burst firing activity in pain function., Results: Under pentobarbital or urethane anesthesia, the patterns of tonic and burst firings were recorded in functionally characterized RT and VPL neurons of Cav3.2 KO mice. Many RT neurons were nociceptive (64% under pentobarbital anesthesia and 50% under urethane anesthesia). Compared to their wild-type (WT) controls, fewer nociceptive RT neurons were found in Cav3.2 KO mice. Both nociceptive and tactile RT neurons showed fewer bursts in Cav3.2 KO mice. Within a burst, RT neurons of Cav3.2 KO mice had a lower spike frequency and less-prominent accelerando-decelerando change. In contrast, VP neurons of Cav3.2 KO mice showed a higher ratio of bursts and a higher discharge rate within a burst than those of the WT control. In addition, the long-lasting tonic firing episodes in RT neurons of the Cav3.2 KO had less stereotypic regularity than their counterparts in WT mice., Conclusions: RT might be important in nociception of the mouse. In addition, we showed an important role of Cav3.2 subtype of T-channel in RT burst firing pattern. The decreased occurrence and slowing of the bursts in RT neurons might cause the increased VP bursts. These changes would be factors contributing to alternation of pain behavior in the Cav3.2 KO mice.

In mammals there are three types of low-voltage-activated (LVA) calcium channels,Cav3.1, Cav3.2 and Cav3.3, which all give rise to T-type Ca2+currents. T-type Ca2+currents have long been known to be highly enriched in a sub-population of medium-sized sensory neurones in the dorsal root ganglia (DRG). However, the identity of the T-type-rich sensory neurones has remained controversial and the precise physiological role of the Cav3.2 calcium channel in these sensory neurones has not been directly addressed. Here we show, using Cav3.2−/− mutant mice,that these channels are essential for the normal temporal coding of moving stimuli by specialized skin mechanoreceptors called D-hair receptors.We show that D-hair receptors from Cav3.2−/− fire approximately 50% fewer spikes in response to ramp-and-hold displacement stimuli compared to wild type receptors. The reduced sensitivity of D-hair receptors in Cav3.2−/− mice is chiefly due to an increase in the mechanical threshold and a substantial temporal delay in the onset of high-frequency firing to moving stimuli.We examined the receptive properties of other cutaneous mechano receptors and Aδ- and C-fibre nociceptors in Cav3.2−/− mice, but found no alteration in their mechanosensitivity compared to Cav3.2+/+mice. However, C-fibre nociceptors recorded in Cav3.2−/− mutant mice displayed a small but statistically significant reduction in their spiking rate during noxious heat ramps when compared to C-fibres in control mice. The T-type calcium channel Cav3.2 is thus not only a highly specific marker of D-hair receptors but is also required to maintain their high sensitivity and above all to ensure ultra rapid temporal detection of skin movement.

T-type calcium channel isoforms expressed in heterologous systems demonstrate marked differences in the biophysical properties of the resulting calcium currents. Such heterogeneity in gating behaviour not only reflects structural differences but is also observed following the regulation of channel activity by a number of ligands. However, the physiological impact of these differences in gating parameters of the T channels has never been evaluated in situ where the unique interplay between T-type calcium and other intrinsic currents is conserved, and T channel activation can be triggered by synaptic stimulation. Here, using the dynamic clamp technique, artificial T conductances were re-incorporated in thalamic neurons devoid of endogenous T currents to dissect the physiological role of the T current gating diversity on neuronal excitability. We demonstrate that the specific kinetics of the T currents in thalamocortical and nucleus reticularis thalami neurons determine the characteristic firing patterns of these neurons. We show that subtle modifications in T channel gating that are at the limit of the resolution achieved in classical biophysical studies in heterologous expression systems have profound consequences for synaptically evoked firing dynamics in native neurons. Moreover, we demonstrate that the biophysical properties of the T current in the voltage region corresponding to the foot of the activation and inactivation curves drastically condition physiologically evoked burst firing with a high degree of synaptic input specificity.

The role of P/Q- and T-type calcium channels in the rhythmic oscillatory behaviour of inferior olive (IO) neurons was investigated in mutant mice. Mice lacking either the CaV2.1 gene of the pore-forming alpha1A subunit for P/Q-type calcium channel, or the CaV3.1 gene of the pore-forming alpha1G subunit for T-type calcium channel were used. In vitro intracellular recording from IO neurons reveals that the amplitude and frequency of sinusoidal subthreshold oscillations (SSTOs) were reduced in the CaV2.1-/- mice. In the CaV3.1-/- mice, IO neurons also showed altered patterns of SSTOs and the probability of SSTO generation was significantly lower (15%, 5 of 34 neurons) than that of wild-type (78%, 31 of 40 neurons) or CaV2.1-/- mice (73%, 22 of 30 neurons). In addition, the low-threshold calcium spike and the sustained endogenous oscillation following rebound potentials were absent in IO neurons from CaV3.1-/- mice. Moreover, the phase-reset dynamics of oscillatory properties of single neurons and neuronal clusters in IO were remarkably altered in both CaV2.1-/- and CaV3.1-/- mice. These results suggest that both alpha1A P/Q- and alpha1G T-type calcium channels are required for the dynamic control of neuronal oscillations in the IO. These findings were supported by results from a mathematical IO neuronal model that incorporated T and P/Q channel kinetics.

The rhythmic motor pathway activation by pacemaker neurons or circuits in the brain has been proposed as the mechanism for the timing of motor coordination, and the abnormal potentiation of this mechanism may lead to a pathological tremor. Here, we show that the potentiation of Ca(V)3.1 T-type Ca(2+) channels in the inferior olive contributes to the onset of the tremor in a pharmacological model of essential tremor. After administration of harmaline, 4- to 10-Hz synchronous neuronal activities arose from the IO and then propagated to cerebellar motor circuits in wild-type mice, but those rhythmic activities were absent in mice lacking Ca(V)3.1 gene. Intracellular recordings in brain-stem slices revealed that the Ca(V)3.1-deficient inferior olive neurons lacked the subthreshold oscillation of membrane potentials and failed to trigger 4- to 10-Hz rhythmic burst discharges in the presence of harmaline. In addition, the selective knockdown of Ca(V)3.1 gene in the inferior olive by shRNA efficiently suppressed the harmaline-induced tremor in wild-type mice. A mathematical model constructed based on data obtained from patch-clamping experiments indicated that harmaline could efficiently potentiate Ca(V)3.1 channels by changing voltage-dependent responsiveness in the hyperpolarizing direction. Thus, Ca(V)3.1 is a molecular pacemaker substrate for intrinsic neuronal oscillations of inferior olive neurons, and the potentiation of this mechanism can be considered as a pathological cause of essential tremor.

In noncontractile cells, increases in intracellular Ca2+ concentration serve as a second messenger to signal proliferation, differentiation, metabolism, motility, and cell death. Many of these Ca2+-dependent regulatory processes operate in cardiomyocytes, although it remains unclear how Ca2+ serves as a second messenger given the high Ca2+ concentrations that control contraction. T-type Ca2+ channels are reexpressed in adult ventricular myocytes during pathologic hypertrophy, although their physiologic function remains unknown. Here we generated cardiac-specific transgenic mice with inducible expression of alpha1G, which generates Cav3.1 current, to investigate whether this type of Ca2+ influx mechanism regulates the cardiac hypertrophic response. Unexpectedly, alpha1G transgenic mice showed no cardiac pathology despite large increases in Ca2+ influx, and they were even partially resistant to pressure overload-, isoproterenol-, and exercise-induced cardiac hypertrophy. Conversely, alpha1G-/- mice displayed enhanced hypertrophic responses following pressure overload or isoproterenol infusion. Enhanced hypertrophy and disease in alpha1G-/- mice was rescued with the alpha1G transgene, demonstrating a myocyte-autonomous requirement of alpha1G for protection. Mechanistically, alpha1G interacted with NOS3, which augmented cGMP-dependent protein kinase type I activity in alpha1G transgenic hearts after pressure overload. Further, the anti-hypertrophic effect of alpha1G overexpression was abrogated by a NOS3 inhibitor and by crossing the mice onto the Nos3-/- background. Thus, cardiac alpha1G reexpression and its associated pool of T-type Ca2+ antagonize cardiac hypertrophy through a NOS3-dependent signaling mechanism.

The epidemics of obesity and metabolic disorders have well-recognized health and economic burdens. Pharmacologic treatments for these diseases remain unsatisfactory with respect to both efficacy and side-effect profiles. Here, we have identified a potential central role for T-type calcium channels in regulating body weight maintenance and sleep. Previously, it was shown that mice lacking CaV3.1 T-type calcium channels have altered sleep/wake activity. We found that these mice were also resistant to high-fat diet-induced weight gain, without changes in food intake or sensitivity to high-fat diet-induced disruptions of diurnal rhythm. Administration of a potent and selective antagonist of T-type calcium channels, TTA-A2, to normal-weight animals prior to the inactive phase acutely increased sleep, decreased body core temperature, and prevented high-fat diet-induced weight gain. Administration of TTA-A2 to obese rodents reduced body weight and fat mass while concurrently increasing lean muscle mass. These effects likely result from better alignment of diurnal feeding patterns with daily changes in circadian physiology and potentially an increased metabolic rate during the active phase. Together, these studies reveal what we believe to be a previously unknown role for T-type calcium channels in the regulation of sleep and weight maintenance and suggest the potential for a novel therapeutic approach to treating obesity.

Voltage-gated T-type Ca(2+) channels (T-channels) are normally expressed during embryonic development in ventricular myocytes but are undetectable in adult ventricular myocytes. Interestingly, T-channels are reexpressed in hypertrophied or failing hearts. It is unclear whether T-channels play a role in the pathogenesis of cardiomyopathy and what the mechanism might be. Here we show that the alpha(1H) voltage-gated T-type Ca(2+) channel (Ca(v)3.2) is involved in the pathogenesis of cardiac hypertrophy via the activation of calcineurin/nuclear factor of activated T cells (NFAT) pathway. Specifically, pressure overload-induced hypertrophy was severely suppressed in mice deficient for Ca(v)3.2 (Ca(v)3.2(-/-)) but not in mice deficient for Ca(v)3.1 (Ca(v)3.1(-/-)). Angiotensin II-induced cardiac hypertrophy was also suppressed in Ca(v)3.2(-/-) mice. Consistent with these findings, cultured neonatal myocytes isolated from Ca(v)3.2(-/-) mice fail to respond hypertrophic stimulation by treatment with angiotensin II. Together, these results demonstrate the importance of Ca(v)3.2 in the development of cardiac hypertrophy both in vitro and in vivo. To test whether Ca(v)3.2 mediates the hypertrophic response through the calcineurin/NFAT pathway, we generated Ca(v)3.2(-/-), NFAT-luciferase reporter mice and showed that NFAT-luciferase reporter activity failed to increase after pressure overload in the Ca(v)3.2(-/-)/NFAT-Luc mice. Our results provide strong genetic evidence that Ca(v)3.2 indeed plays a pivotal role in the induction of calcineurin/NFAT hypertrophic signaling and is crucial for the activation of pathological cardiac hypertrophy.

We previously have shown in mice and rats, enhanced leukocyte recruitment to airway postcapillary venules after excessive distention imposed by the application of positive end-expiratory pressure. Because P-selectin was shown to be essential for this outcome, we sought to establish an in vitro endothelial cell model and determine the mechanisms whereby mechanical distension alters adhesion molecule expression. P-selectin surface expression on mouse jugular vein endothelial cells exposed to cyclic stretch (5 or 20% elongation for 5 min; Flexercell) were compared with static cells. The larger, pathophysiological regimen of cyclic stretch showed a 54% increase in P-selectin expression after stretch compared with static cells. This response was attenuated but confirmed in tracheal venular endothelium (29% increase). We questioned whether these changes were dependent on increases in intracellular Ca(2+) through voltage-gated Ca(2+) channels. The stretch-induced increase in P-selectin expression was substantially decreased by pretreatment with the T-type Ca(2+) channel inhibitor mibefradil (76% inhibition). Furthermore, when the Ca(v)3.1 T-type Ca(2+) channel expression was decreased in both endothelial cell subtypes with specific small-interfering RNA, the distension-induced expression of P-selectin decreased to levels less than that observed in static cells. We conclude that P-selectin expression on systemic venular endothelial cells contributes to a proinflammatory phenotype after mechanical stretch and can be selectively modulated by voltage-gated calcium channel inhibition.

To assess the role of alpha(1G) T-type Ca2+ channels in neuropathic pain after L5 spinal nerve ligation, we examined behavioral pain susceptibility in mice lacking CaV3.1 (alpha1G(-/-)), the gene encoding the pore-forming units of these channels. Reduced spontaneous pain responses and an increased threshold for paw withdrawal in response to mechanical stimulation were observed in these mice. The alpha1G(-/-) mice also showed attenuated thermal hyperalgesia in response to both low-(IR30) and high-intensity (IR60) infrared stimulation. Our results reveal the importance of alpha(1G) T-type Ca2+ channels in the development of neuropathic pain, and suggest that selective modulation of alpha1G subtype channels may provide a novel approach to the treatment of allodynia and hyperalgesia.

In addition to its inhibitory action, reports have shown that, in sensory neurons, GABA can be responsible for excitatory effects leading to painful behavior. The cellular mechanisms for these excitatory effects remain largely unknown. Although the high intracellular chloride concentration allows GABA(A) receptor activation to depolarize all adult sensory neurons, we show that GABA, acting through GABA(A) receptors, can generate, in vitro, action potential and intracellular Ca(2+) increase only in a subset of neurons expressing a prominent T-type Ca(2+) current. When recorded from Cav3.2(-/-) mice, T-type Ca(2+) current was totally abolished in this morphologically identified subset of neurons and GABA(A) receptors activation did not induce electrical activity nor intracellular Ca(2+) increase. In addition to gene inhibition, pharmacological analysis of Ca(2+) channel subunits shows the amplifying role of T-current in GABA(A) current-induced membrane depolarization and the involvement of both T-current and high voltage activated Ca(2+) current in GABA(A)-induced intracellular Ca(2+) increase. Altogether, these data establish that the Cav3.2/alpha1H, T-current is responsible for GABA-induced cell excitability and intracellular Ca(2+) increase. Our results reveal a positive cross-talk between T-channel and GABA(A) receptor in adult sensory neurons and indicate that Cav3.2/alpha1H, T-type Ca(2+) channel may be the molecular determinant for excitatory effects of GABA in peripheral somatosensory system.

In spermatozoa, voltage-dependent calcium channels (VDCC) have been involved in different cellular functions like acrosome reaction (AR) and sperm motility. Multiple types of VDCC are present and their relative contribution is still a matter of debate. Based mostly on pharmacological studies, low-voltage-activated calcium channels (LVA-CC), responsible of the inward current in spermatocytes, were described as essential for AR in sperm. The development of Ca(V)3.1 or Ca(V)3.2 null mice provided the opportunity to evaluate the involvement of such LVA-CC in AR and sperm motility, independently of pharmacological tools. The inward current was fully abolished in spermatogenic cells from Ca(V)3.2 deficient mice. This current is thus only due to Ca(V)3.2 channels. We showed that Ca(V)3.2 channels were maintained in sperm by Western-blot and immunohistochemistry experiments. Calcium imaging experiments revealed that calcium influx in response to KCl was reduced in Ca(V)3.2 null sperm in comparison to control cells, demonstrating that Ca(V)3.2 channels were functional. On the other hand, no difference was noticed in calcium signaling induced by zona pellucida. Moreover, neither biochemical nor functional experiments, suggested the presence of Ca(V)3.1 channels in sperm. Despite the Ca(V)3.2 channels contribution in KCl-induced calcium influx, the reproduction parameters remained intact in Ca(V)3.2 deficient mice. These data demonstrate that in sperm, besides Ca(V)3.2 channels, other types of VDCC are activated during the voltage-dependent calcium influx of AR, these channels likely belonging to high-voltage activated Ca(2+) channels family. The conclusion is that voltage-dependent calcium influx during AR is due to the opening of redundant families of calcium channels.

In response to light, the mouse retinal pigment epithelium (RPE) generates a series of slow changes in potential that are referred to as the c-wave, fast oscillation (FO), and light peak (LP) of the electroretinogram (ERG). The LP is generated by a depolarization of the basolateral RPE plasma membrane by the activation of a calcium-sensitive chloride conductance. We have previously shown that the LP is reduced in both mice and rats by nimodipine, which blocks voltage-dependent calcium channels (VDCCs) and is abnormal in lethargic mice, carrying a null mutation in the calcium channel beta(4) subunit. To define the alpha(1) subunit involved in this process, we examined mice lacking Ca(V)1.3. In comparison with wild-type (WT) control littermates, LPs were reduced in Ca(V)1.3(-/-) mice. This pattern matched closely with that previously noted in lethargic mice, confirming a role for VDCCs in regulating the signaling pathway that culminates in LP generation. These abnormalities do not reflect a defect in rod photoreceptor activity, which provides the input to the RPE to generate the c-wave, FO, and LP, because ERG a-waves were comparable in WT and Ca(V)1.3(-/-) littermates. Our results identify Ca(V)1.3 as the principal pore-forming subunit of VDCCs involved in stimulating the ERG LP.

Despite their presence in many tissues and their potential implication in various disease states, low-voltage activated T-type calcium channels (T-channels) have only recently become targets of interest. Unfortunately, the lack of selective T-channel blockers has hampered further characterisation of these channels. The recent availability of cloned T-channels, the Ca(V)3 proteins, facilitates identification of novel T-channel blockers. Also, studies performed in knockout animals have fostered novel interest. Selective inhibition of T-channels may have clinical importance in cardiovascular diseases, some forms of epilepsy, sleep disorders, pain and possibly cancer. This review focuses on novel research approaches to discover potent and selective T-channel modulators. These molecules may be potential drugs for treating human diseases, as well as important tools to decipher the physiological role of these channels.

Sleep is characterized by synchronized electrical activities of the thalamocortical network, which can be identified as the EEG oscillations during sleep. T-type calcium channels have been implicated in the occurrence of sleep waves, and burst firings in the thalamic neurons driven by these channels are known to be essential for modulation of sleep rhythms. Studies showed that alpha1(G) T-type calcium channel knockout mice had defects in sleep waves such as lack of delta oscillations (1-4 Hz) and alteration of sleep spindles (7-15 Hz), which are known to be modulated by T-currents in the thalamus. The mutation also affected the sleep-wake transition, thus resulting in decreased NREM sleep and increased sleep disturbance. These findings support the idea that alpha1(G) T-type calcium channels contribute to sleep waves as well as to behavioral state of sleep.

The generation of the mammalian heartbeat is a complex and vital function requiring multiple and coordinated ionic channel activities. The functional role of low-voltage activated (LVA) T-type calcium channels in the pacemaker activity of the sinoatrial node (SAN) is, to date, unresolved. Here we show that disruption of the gene coding for CaV3.1/alpha1G T-type calcium channels (cacna1g) abolishes T-type calcium current (I(Ca,T)) in isolated cells from the SAN and the atrioventricular node without affecting the L-type Ca2+ current (I(Ca,L)). By using telemetric electrocardiograms on unrestrained mice and intracardiac recordings, we find that cacna1g inactivation causes bradycardia and delays atrioventricular conduction without affecting the excitability of the right atrium. Consistently, no I(Ca,T) was detected in right atrium myocytes in both wild-type and CaV3.1(-/-) mice. Furthermore, inactivation of cacna1g significantly slowed the intrinsic in vivo heart rate, prolonged the SAN recovery time, and slowed pacemaker activity of individual SAN cells through a reduction of the slope of the diastolic depolarization. Our results demonstrate that CaV3.1/T-type Ca2+ channels contribute to SAN pacemaker activity and atrioventricular conduction.

It has long been suspected that sensory signal transmission is inhibited in the mammalian brain during sleep. We hypothesized that Cav3.1 T-type Ca2+ channel currents inhibit thalamic sensory transmission to promote sleep. We found that T-type Ca2+ channel activation caused prolonged inhibition (>9 s) of action-potential firing in thalamic projection neurons of WT but not Cav3.1 knockout mice. Inhibition occurred with synaptic transmission blocked and required an increase of intracellular Ca2+. Furthermore, focal deletion of the gene encoding Cav3.1 from the rostral-midline thalamus by using Cre/loxP recombination led to frequent and prolonged arousal, which fragmented and reduced sleep. Interestingly, sleep was not disturbed when Cav3.1 was deleted from cortical pyramidal neurons. These findings support the hypothesis that thalamic T-type Ca2+ channels are required to block transmission of arousal signals through the thalamus and to stabilize sleep.

Mammalian acrosome reaction (AR) requires successive activation of three different types of calcium channels (T-type channels, Inositol-3-phosphate (InsP3) receptors, and TRPC2 channels). All the calcium signaling is under the control of the activation of the first-one, a T-type calcium channel. The molecular characterization of the T-type calcium channel is still a matter of debate, previous reports showing the presence of transcripts for Ca(V)3.1 and Ca(V)3.2 subunits. Using mice deficient for Ca(V)3.1 subunit, we show that the T-type current density in spermatogenic cells is not reduced in deficient mice versus control mice. We characterized the biophysical and pharmacological properties of T-type current in spermatogenic cells from Ca(V)3.1 deficient mice. Biophysical and pharmacological properties of spermatogenic T-type current from wild-type and Ca(V)3.1 deficient mice demonstrate that Ca(V)3.3 does not contribute to T-type current. Moreover, nickel and amiloride inhibit T-type currents in deficient and wild-type mice with similar potencies. These results demonstrate that T-type currents in spermatogenic cells is due to Ca(V)3.2 subunit and that Ca(V)3.1 contributes to a very negligible extent to the T-type currents. Thus, the deficient Ca(V)3.1 mouse model allows the characterization of native Ca(V)3.2 currents in spermatogenic cells. Spermatogenic Ca(V)3.2 currents present specific feature in comparison to the cloned Ca(V)3.2 current so far. More particularly, the time-dependence of recovery from short-term inactivation of native spermatogenic Ca(V)3.2 is close to 100 millisecond, a value expected for Ca(V)3.1 current., (Copyright 2003 Wiley-Liss, Inc.)

Alterations in thalamic T-type Ca2+ channels are thought to contribute to the pathogenesis of absence seizures. Here, we found that mice with a null mutation for the pore-forming alpha1A subunits of P/Q-type channels (alpha1A-/- mice) were prone to absence seizures characterized by typical spike-and-wave discharges (SWDs) and behavioral arrests. Isolated thalamocortical relay (TC) neurons from these mice showed increased T-type Ca2+ currents in vitro. To examine the role of increased T-currents in alpha1A-/- TC neurons, we cross-bred alpha1A-/- mice with mice harboring a null mutation for the gene encoding alpha1G, a major isotype of T-type Ca2+ channels in TC neurons. alpha1A-/-/alpha1G-/- mice showed a complete loss of T-type Ca2+ currents in TC neurons and displayed no SWDs. Interestingly, alpha1A-/-/alpha1G+/- mice had 75% of the T-type Ca2+ currents in TC neurons observed in alpha1A+/+/alpha1G+/+ mice and showed SWD activity that was quantitatively similar to that in alpha1A-/-/alpha1G+/+ mice. Similar results were obtained using double-mutant mice harboring the alpha1G mutation plus another mutation also used as a model for absence seizures, i.e., lethargic (beta4(lh/lh)), tottering (alpha1A(tg/tg)), or stargazer (gamma2(stg/stg)). The present results reveal that alpha1G T-type Ca2+ channels play a critical role in the genesis of spontaneous absence seizures resulting from hypofunctioning P/Q-type channels, but that the augmentation of thalamic T-type Ca2+ currents is not an essential step in the genesis of absence seizures.