Modulation Of T-type Calcium Channels Research Articles

Protein kinase C epsilon-mediated modulation of T-type calcium channels underlies alcohol withdrawal hyperexcitability in the midline thalamus.

Millions of people struggle with alcohol use disorder (AUD). Abrupt abstinence after a period of chronic alcohol use can precipitate the alcohol withdrawal syndrome (AWS), which includes hyperexcitability and, potentially, seizures. We have shown that T-type Ca2+ channels are novel, sensitive targets of alcohol, an effect that is dependent upon protein kinase C (PKC). The purpose of this study was to (1) understand midline thalamic neuronal hyperexcitability during alcohol withdrawal and its dependence on PKC; (2) characterize T channel functional changes using both current clamp and voltage clamp methods; and (3) determine which PKC isoform may be responsible for alcohol withdrawal (WD) effects. Whole-cell patch clamp recordings were performed in midline thalamic neurons in brain slices prepared from C57bl/6 mice that underwent chronic intermittent alcohol exposure in a standard vapor chamber model. The recordings were compared to those from air-exposed controls. T-channel inactivation curves and burst responses were acquired through voltage-clamp and current-clamp recordings, respectively. Whole-cell voltage clamp recordings of native T-type current exhibited a depolarizing shift in the voltage-dependency of inactivation during alcohol withdrawal compared to air-exposed controls. A PKCε translocation inhibitor peptide mitigated this change. Current clamp recordings demonstrated more spikes per burst during alcohol withdrawal. Consistent with voltage clamp findings, the PKCɛ translocation inhibitor peptide reduced the number of spikes per burst after WD. We found that alcohol WD produces T channel-mediated hyperexcitability in the midline thalamus, produced in part by a shift in the inactivation curve consistent with greater availability of T current. WD effects on T current inactivation were reduced to control levels by blocking PKCε translocation. Our results demonstrate that PKCε translocation plays an important role in the regulation of alcohol withdrawal-induced hyperexcitability in midline thalamic circuitry.

Inhibition of human recombinant T-type calcium channels by phytocannabinoids in vitro.

T-type Ca channels (ICa ) regulate neuronal excitability and contribute to neurotransmitter release. The phytocannabinoids Δ9 -tetrahydrocannabinol and cannabidiol effectively modulate T-type ICa , but effects of other biologically active phytocannabinoids on these channels are unknown. We thus investigated the modulation of T-type ICa by low abundance phytocannabinoids. A fluorometric (fluorescence imaging plate reader [FLIPR]) assay was used to investigate modulation of human T-type ICa (CaV 3.1, 3.2 and 3.3) stably expressed in FlpIn-TREx HEK293 cells. The biophysical effects of some compounds were examined using whole-cell patch clamp recordings. In the FLIPR assay, all 11 phytocannabinoids tested modulated T-type ICa , with most inhibiting CaV 3.1 and CaV 3.2 more effectively than CaV 3.3. Cannabigerolic acid was the most potent inhibitor of CaV 3.1 (pIC50 6.1±0.6) and CaV 3.2 (pIC50 6.4±0.4); in all cases, phytocannabinoid acids were more potent than their corresponding neutral forms. In patch clamp recordings, cannabigerolic acid inhibited CaV 3.1 and 3.2 with similar potency to the FLIPR assay; the inhibition was associated with significant hyperpolarizing shift in activation and steady-state inactivation of these channels. In contrast, cannabidiol, cannabidivarin, and cannabigerol only affected channel inactivation. Modulation of T-type calcium channels is a common property of phytocannabinoids, which all increase steady-state inactivation at physiological membrane potentials, with some also affecting channel activation. Thus, T-type ICa may be a common site of action for phytocannabinoids, and the diverse actions of phytocannabinoids on channel gating may provide insight into structural requirement for selective T-type ICa modulators.

A modulator of the low-voltage-activated T-type calcium channel that reverses HIV glycoprotein 120-, paclitaxel-, and spinal nerve ligation-induced peripheral neuropathies.

The voltage-gated calcium channels CaV3.1-3.3 constitute the T-type subfamily, whose dysfunctions are associated with epilepsy, psychiatric disorders, and chronic pain. The unique properties of low-voltage-activation, faster inactivation, and slower deactivation of these channels support their role in modulation of cellular excitability and low-threshold firing. Thus, selective T-type calcium channel antagonists are highly sought after. Here, we explored Ugi-azide multicomponent reaction products to identify compounds targeting T-type calcium channel. Of the 46 compounds tested, an analog of benzimidazolonepiperidine-5bk (1-{1-[(R)-{1-[(1S)-1-phenylethyl]-1H-1,2,3,4-tetrazol-5-yl}(thiophen-3-yl)methyl]piperidin-4-yl}-2,3-dihydro-1H-1,3-benzodiazol-2-one) modulated depolarization-induced calcium influx in rat sensory neurons. Modulation of T-type calcium channels by 5bk was further confirmed in whole-cell patch clamp assays in dorsal root ganglion (DRG) neurons, where pharmacological isolation of T-type currents led to a time- and concentration-dependent regulation with a low micromolar IC50. Lack of an acute effect of 5bk argues against a direct action on T-type channels. Genetic knockdown revealed CaV3.2 to be the isoform preferentially modulated by 5bk. High voltage-gated calcium, as well as tetrodotoxin-sensitive and -resistant sodium, channels were unaffected by 5bk. 5bk inhibited spontaneous excitatory postsynaptic currents and depolarization-evoked release of calcitonin gene-related peptide from lumbar spinal cord slices. Notably, 5bk did not bind human mu, delta, or kappa opioid receptors. 5bk reversed mechanical allodynia in rat models of HIV-associated neuropathy, chemotherapy-induced peripheral neuropathy, and spinal nerve ligation-induced neuropathy, without effects on locomotion or anxiety. Thus, 5bk represents a novel T-type modulator that could be used to develop nonaddictive pain therapeutics.

Dual role of T-type calcium channels in anxiety-related behavior.

T-type calcium channels are low voltage activated calcium channels that are widely expressed in various brain regions including stress-responsive regions. These channels regulate the diverse functions of the central nervous system, and modulation of these channels is shown to modulate the anxiety. Studies have described that modulation of T-type calcium channels may either aggravate or ameliorate anxiety-related behavior, suggesting the dual role of these channels. The studies employing animals with overexpression of T-type calcium channels reported their anxiety-inducing role. Therefore, the blockade of these channels using various pharmacological agents such as ethosuximide, plant extracts of linalool or rosemary, and corticotropin-releasing factor (CRF) is reported to ameliorate anxiety. On the contrary, knockout of the gene encoding these channels predisposes the rodents to anxiety-related disorders, suggesting the anxiety-attenuating role of these channels. It may be possible that these channels in normal or basal state attenuate anxiety, whereas activation of these channels in stressful condition may produce anxiety. The present review describes the dual role of T-type calcium channels in anxiety-related behavior in both preclinical and clinical studies.

Locus coeruleus activation enhances thalamic feature selectivity via norepinephrine regulation of intrathalamic circuit dynamics.

We investigated locus coeruleus (LC) modulation of thalamic feature selectivity through reverse correlation analysis of single-unit recordings from different stages of the rat vibrissa pathway. LC activation increased feature selectivity, drastically improving thalamic information transmission. We found this improvement was dependent on both local activation of α-adrenergic receptors and modulation of T-type calcium channels in the thalamus and was not due to LC modulation of trigeminothalamic feedforward or corticothalamic feedback inputs. Tonic spikes with LC stimulation carried 3-times the information than did tonic spikes without LC stimulation. Modelling confirmed norepinephrine (NE) regulation of intrathalamic circuit dynamics led to the improved information transmission. Behavioral data demonstrated that LC activation increased the perceptual performance of animals performing tactile discrimination tasks through LC-NE optimization of thalamic sensory processing. These results suggest a new sub-dimension within the tonic mode in which brain state can optimize thalamic sensory processing through modulation of intrathalamic circuit dynamics.

Synthesis and biological evaluation of pyrrolidine-based T-type calcium channel inhibitors for the treatment of neuropathic pain

The treatment of neuropathic pain is one of the urgent unmet medical needs and T-type calcium channels are promising therapeutic targets for neuropathic pain. Several potent T-type channel inhibitors showed promising in vivo efficacy in neuropathic pain animal models and are being investigated in clinical trials. Herein we report development of novel pyrrolidine-based T-type calcium channel inhibitors by pharmacophore mapping and structural hybridisation followed by evaluation of their Cav3.1 and Cav3.2 channel inhibitory activities. Among potent inhibitors against both Cav3.1 and Cav3.2 channels, a promising compound 20n based on in vitro ADME properties displayed satisfactory plasma and brain exposure in rats according to in vivo pharmacokinetic studies. We further demonstrated that 20n effectively improved the symptoms of neuropathic pain in both SNL and STZ neuropathic pain animal models, suggesting modulation of T-type calcium channels can be a promising therapeutic strategy for the treatment of neuropathic pain.

GABAB receptors suppress burst-firing in reticular thalamic neurons

ABSTRACTBurst-firing in thalamic neurons is known to play a key role in mediating thalamocortical (TC) oscillations that are associated with non-REM sleep and some types of epileptic seizure. Within the TC system the primary output of GABAergic neurons in the reticular thalamic nucleus (RTN) is thought to induce the de-inactivation of T-type calcium channels in thalamic relay (TR) neurons, promoting burst-firing drive to the cortex and the propagation of TC network activity. However, RTN neurons also project back onto other neurons within the RTN. The role of this putative negative feedback upon the RTN itself is less well understood, although is hypothesized to induce de-synchronization of RTN neuron firing leading to the suppression of TC oscillations. Here we tested two hypotheses concerning possible mechanisms underlying TC oscillation modulation. Firstly, we assessed the burst-firing behavior of RTN neurons in response to GABAB receptor activation using acute brain slices. The selective GABAB receptor agonist baclofen was found to induce suppression of burst-firing concurrent with effects on membrane input resistance. Secondly, RTN neurons express CaV3.2 and CaV3.3 T-type calcium channel isoforms known to contribute toward TC burst-firing and we examined the modulation of these channels by GABAB receptor activation. Utilizing exogenously expressed T-type channels we assessed whether GABAB receptor activation could directly alter T-type calcium channel properties. Overall, GABAB receptor activation had only modest effects on CaV3.2 and CaV3.3 isoforms. The only effect that could be predicted to suppress burst-firing was a hyperpolarized shift in the voltage-dependence of inactivation, potentially causing lower channel availability at membrane potentials critical for burst-firing. Conversely, other effects observed such as a hyperpolarized shift in the voltage-dependence of activation of both CaV3.2 and CaV3.3 as well as increased time constant of activation of the CaV3.3 isoform would be expected to enhance burst-firing. Together, we hypothesize that GABAB receptor activation mediates multiple downstream effectors that combined act to suppress burst-firing within the RTN. It appears unlikely that direct GABAB receptor-mediated modulation of T-type calcium channels is the major mechanistic contributor to this suppression.

Modulation of T-type calcium channels by bioactive lipids

T-type calcium channels (T-channels/CaV3) have unique biophysical properties allowing a calcium influx at resting membrane potential of most cells. T-channels are ubiquitously expressed in many tissues and contribute to low-threshold spikes and burst firing in central neurons as well as to pacemaker activities in cardiac cells. They also emerged as potential targets to treat cancer and hypertension. Regulation of these channels appears complex, and several studies have indicated that CaV3.1, CaV3.2, and CaV3.3 currents are directly inhibited by multiple endogenous lipids independently of membrane receptors or intracellular pathways. These bioactive lipids include arachidonic acid and ω3 poly-unsaturated fatty acids; the endocannabinoid anandamide and other N-acylethanolamides; the lipoamino-acids and lipo-neurotransmitters; the P450 epoxygenase metabolite 5,6-epoxyeicosatrienoic acid; as well as similar molecules with 18-22 carbons in the alkyl chain. In this review, we summarize evidence for direct effects of these signaling molecules, the molecular mechanisms underlying the current inhibition, and the involved chemical features. The impact of this modulation in physiology and pathophysiology is discussed with a special emphasis on pain aspects and vasodilation. Overall, these data clearly indicate that T-current inhibition is an important mechanism by which bioactive lipids mediate their physiological functions.

Molecular pathways underlying the modulation of T-type calcium channels by neurotransmitters and hormones

Low-voltage-activated T-type calcium channels are expressed in various tissues, especially in the brain, where they promote neuronal firing and are involved in slow wave sleep and absence epilepsy. While the transduction pathways by which hormones and neurotransmitters modulate high-voltage-activated calcium channels are beginning to be unraveled, those implicated in T-type calcium channel regulation remain obscure. Several neurotransmitters and hormones regulate native T-type calcium channels, although some contradictory data have been reported depending on the cell type studied. This review focuses on the short-term (minutes range) modulation of T-type calcium channels by neurotransmitters and hormones and on the roles of G proteins and protein kinases in these modulatory effects. Results obtained in different native tissues are discussed and compared with the more recent studies of the three cloned T-type calcium channels Ca V3.1, Ca V3.2 and Ca V3.3 in expression systems.

The relevance of T-type calcium antagonists: a profile of mibefradil.

L- and T-type voltage-dependent transmembrane calcium channels are important for normal functioning of the cardiovascular system. T-type channels are a heterogeneous group, and have physiologic and pathophysiologic relevance in a number of organ systems, including the heart and central nervous system. They appear to be involved in the control of blood pressure in patients with essential hypertension and in protection from ischemic damage. Alterations of both L- and T-type calcium channels are involved in the development of hypertension. Pharmacologic modulation of T-type calcium channels appears to reduce membrane calcium flux and ameliorate hypertension. During early ischemic damage, T-type calcium channels appear to remain functional whereas L-type channels are already inactivated. T-type calcium channels also appear to be involved in the development of supraventricular arrhythmias, some forms of arrhythmias in cardiomyopathy, and cardiac hypertrophy. The heterogeneity of T-type calcium channels should make it possible to target drugs to specific subgroups of T-type calcium channels. A new class of calcium antagonist, the benzimidazolyl-substituted tetraline derivatives, has been shown to block both L- and T-type calcium channels. The first member of this class approved for clinical use is mibefradil. Clinical studies have demonstrated the efficacy of mibefradil in lowering blood pressure and as an antianginal and antiischemic agent. At clinically recommended doses, mibefradil has a heart rate lowering effect without a negative inotropic effect, and a favorable side effect profile. Because it is metabolized by the cytochrome P450 pathway, it should be used cautiously with other agents similarly metabolized.

Voltage-dependent modulation of T-type calcium channels by protein tyrosine phosphorylation.

A T-type Ca2+ channel is expressed during differentiation of the male germ lineage in the mouse and is retained in sperm, where is it activated by contact with the the egg's extracellular matrix and controls sperm acrosomal exocytosis. Here, we examine the regulation of this Ca2+ channel in dissociated spermatogenic cells from the mouse using the whole-cell patch-clamp technique. T currents were enhanced, or facilitated, after strong depolarizations or high frequency stimulation. Voltage-dependent facilitation increased the Ca2+ current by an average of 50%. The same facilitation is produced by antagonists of protein tyrosine kinase activity. Conversely, antagonists of tyrosine phosphatase activity block voltage-dependent facilitation of the current. These data are consistent with the presence of a two-state model, in which T channels are maintained in a low (or zero) conductance state by tonic tyrosine phosphorylation and can be activated to a high conductance state by a tyrosine phosphatase activity. The positive and negative modulation of this channel by the tyrosine phosphorylation state provides a plausible mechanism for the control of sperm activity during the early stages of mammalian fertilization.