Molecular biology of T-type calcium channels.

This review summarizes recent progress on the molecular biology of low voltage-gated, T-type, calcium channels. The genes encoding these channels were identified by molecular cloning of cDNAs that were similar in sequence to the alpha1 subunit of high voltage-activated Ca2+ channels. Three T-channel genes were identified: CACNA1G, encoding Cav3.1; CACNA1H, encoding Cav3.2; and CACNA1I, encoding Cav3.3. Recent studies have focused on how these genes give rise to alternatively spliced transcripts, and how this splicing affects channel activity. A second area of focus is on how single nucleotide polymorphisms (SNPs) alter channel activity. Based on their distribution in thalamic nuclei, coupled with the physiological role they play in thalamic oscillations, leads to the conclusion that SNPs in T-channel genes may contribute to neurological disorders characterized by thalamocortical dysrhythmia, such as generalized epilepsy.

The role of T-type calcium channels in epilepsy and pain.

T-type calcium channels open in response to small depolarizations of the plasma membrane. The entry of two positive charges with every calcium ion leads to a further depolarization of the membrane, the low threshold spike, and opening of channels that have a higher threshold. In this manner, T-channels play an important pacemaker role in gating the activity of Na+ and Ca2+ channels. T-channels are preferentially expressed in dendrites, suggesting they play important roles in synaptic integration. Pharmacological evidence indicates that they are expressed in the receptive fields of sensory neurons, suggesting they play a primary role in nociception. Molecular cloning of the three T-channel genes has allowed detailed studies on their channel properties, pharmacology, distribution in the brain, up-regulation in animal models of disease, and provided the tools to screen for novel drugs. Studies on transgenic animals have provided the proof-of-concept that T-channels are important drug targets for the treatment of absence epilepsy and neuropathic pain. Mutations in ion channel genes, or channelopathies, have been found in many diseases. Similarly, T-channel gene mutations have been found in patients with childhood absence epilepsy. Considering the important role T-channels play in the thalamus, it is likely that T-channel mutations also contribute to a wider range of disorders characterized by thalamocortical dysrhythmia.

Molecular characterization of T-type calcium channels.

Molecular cloning of the low voltage-gated, T-type, calcium channel family opened new avenues of research into their structure-function, distribution, pharmacology, and regulation. Cloning of mammalian cDNAs led to the identification of three T-channel genes: CACNA1G, encoding Cav3.1; CACNA1H, encoding Cav3.2; and CACNA1I, encoding Cav3.3. This allowed sequencing of these genes in absence epilepsy patients, and the identification of single nucleotide polymorphisms (SNPs) that alter channel activity. Their distribution in thalamic nuclei, coupled with the physiological role they play in thalamic oscillations, leads to the conclusion that SNPs in T-channel genes may contribute to neurological disorders characterized by thalamocortical dysrhythmia, such as generalized epilepsy. This section reviews the structure of T-channels, how splicing affects structure and function, how SNPs alter channel activity, and how high voltage-activated auxiliary subunits affect T-channels.

T-type Ca2+ channels encode prior neuronal activity as modulated recovery rates.

T-type Ca2+ channels give rise to low-threshold inward currents that are central determinants of neuronal excitability. The availability of T-type Ca2+ channels is strongly influenced by voltage-dependent inactivation and recovery from inactivation. Here, we show that native and cloned T-type Ca2+ channel subunits selectively encode specific aspects of prior membrane potential changes via a powerful modulation of the rates with which these channels recover from inactivation. Increasing the duration of subthreshold (-70 to -55 mV) conditioning depolarizations caused a pronounced slowing of subsequent recovery from inactivation of both cloned (Ca(v)3.1-3.3) and native T-type channels (thalamic neurones). The scaling of recovery rates with increasing duration of conditioning depolarizations could be well described by a power law function. Different T-type channel isoforms exhibited overlapping but complementary ranges of recovery rates. Intriguingly, scaling of recovery rates was dramatically reduced in Ca(v)3.2 and Ca(v)3.3, but not Ca(v)3.1 subunits, when mock action potentials were superimposed on conditioning depolarizations. Our results suggest that different T-type channel subunits exhibit dramatic differences in scaling relationships, in addition to well-described differences in other biophysical properties. Furthermore, the availability of T-type channels is powerfully modulated over time, depending on the patterns of prior activity that these channels have encountered. These data provide a novel mechanism for cellular short-term plasticity on the millisecond to second time scale that relies on biophysical properties of specific T-type Ca2+ channel subunits.

Direct inhibition of T-type calcium channels by the endogenous cannabinoid anandamide.

Low-voltage-activated or T-type Ca(2+) channels (T-channels) are widely expressed, especially in the central nervous system where they contribute to pacemaker activities and are involved in the pathogenesis of epilepsy. Proper elucidation of their cellular functions has been hampered by the lack of selective pharmacology as well as the absence of generic endogenous regulations. We report here that both cloned (alpha(1G), alpha(1H) and alpha(1I) subunits) and native T-channels are blocked by the endogenous cannabinoid, anandamide. Anandamide, known to exert its physiological effects through cannabinoid receptors, inhibits T-currents independently from the activation of CB1/CB2 receptors, G-proteins, phospholipases and protein kinase pathways. Anandamide appears to be the first endogenous ligand acting directly on T-channels at submicromolar concentrations. Block of anandamide membrane transport by AM404 prevents T-current inhibition, suggesting that anandamide acts intracellularly. Anandamide preferentially binds and stabilizes T-channels in the inactivated state and is responsible for a significant decrease of T-currents associated with neuronal firing activities. Our data demonstrate that anandamide inhibition of T-channels can regulate neuronal excitability and account for CB receptor-independent effects of this signaling molecule.

Gating kinetics of the alpha1I T-type calcium channel.

The alpha1I T-type calcium channel inactivates almost 10-fold more slowly than the other family members (alpha1G and alpha1H) or most native T-channels. We have examined the underlying mechanisms using whole-cell recordings from rat alpha1I stably expressed in HEK293 cells. We found several kinetic differences between alpha1G and alpha1I, including some properties that at first appear qualitatively different. Notably, alpha1I tail currents require two or even three exponentials, whereas alpha1G tails were well described by a single exponential over a wide voltage range. Also, closed-state inactivation is more significant for alpha1I, even for relatively strong depolarizations. Despite these differences, gating of alpha1I can be described by the same kinetic scheme used for alpha1G, where voltage sensor movement is allosterically coupled to inactivation. Nearly all of the rate constants in the model are 5-12-fold slower for alpha1I, but the microscopic rate for channel closing is fourfold faster. This suggests that T-channels share a common gating mechanism, but with considerable quantitative variability.

Inactivation determinants in segment IIIS6 of Ca(v)3.1.

1. Low threshold, T-type, Ca(2+) channels of the Ca(v)3 family display the fastest inactivation kinetics among all voltage-gated Ca(2+) channels. The molecular inactivation determinants of this channel family are largely unknown. Here we investigate whether segment IIIS6 plays a role in Ca(v)3.1 inactivation as observed previously in high voltage-activated Ca(2+) channels. 2. Amino acids that are identical in IIIS6 segments of all Ca(2+) channel subtypes were mutated to alanine (F1505A, F1506A, N1509A, F1511A, V1512A, F1519A, FV1511/1512AA). Additionally M1510 was mutated to isoleucine and alanine. 3. The kinetic properties of the mutants were analysed with the two-microelectrode voltage-clamp technique after expression in Xenopus oocytes. The time constant for the barium current (I(Ba)) inactivation, tau(inact), of wild-type channels at -20 mV was 9.5 +/- 0.4 ms; the corresponding time constants of the mutants ranged from 9.2 +/- 0.4 ms in V1512A to 45.7 +/- 5.2 ms (4.8-fold slowing) in M1510I. Recovery at -80 mV was most significantly slowed by V1512A and accelerated by F1511A. 4. We conclude that amino acids M1510, F1511 and V1512 corresponding to previously identified inactivation determinants in IIIS6 of Ca(v)2.1 (Hering et al. 1998) have a significant role in Ca(v)3.1 inactivation. These data suggest common elements in the molecular architecture of the inactivation mechanism in high and low threshold Ca(2+) channels.

Block of cloned human T-type calcium channels by succinimide antiepileptic drugs.

Inhibition of T-type Ca(2+) channels has been proposed to play a role in the therapeutic action of succinimide antiepileptic drugs. Despite the widespread acceptance of this hypothesis, recent studies using rat and cat neurons have failed to confirm inhibition of T-type currents at therapeutically relevant concentrations. The present study re-examines this issue using the three cloned human channels that constitute the T-type family: alpha 1G, alpha 1H, and alpha 1I. The cloned cDNAs were stably transfected and expressed into mammalian cells, leading to the appearance of typical T-type currents. The results demonstrate that both ethosuximide and the active metabolite of methsuximide, alpha-methyl-alpha-phenylsuccinimide (MPS), block human T-type channels in a state-dependent manner, with higher affinity for inactivated channels. In contrast, succinimide analogs that are not anticonvulsive were relatively poor blockers. The apparent affinity of MPS for inactivated states of the three channels was estimated using two independent measures: K(I) for alpha 1G and alpha 1I was 0.3 to 0.5 mM and for alpha 1H was 0.6 to 1.2 mM. T-type channels display current at the end of long pulses (persistent current), and this current was especially sensitive to block (ethosuximide IC(50) = 0.6 mM). These drugs also reduced both the size of the T-type window current region and the currents elicited by a mock low threshold spike. We conclude that succinimide antiepileptic drugs are capable of blocking human T-type channels at therapeutically relevant concentrations.

Ca(v)3.2 channel is a molecular substrate for inhibition of T-type calcium currents in rat sensory neurons by nitrous oxide.

Although nitrous oxide (N(2)O; laughing gas) remains widely used as an anesthetic and analgesic in clinical practice, its cellular mechanisms of action remain inadequately understood. In this report, we examined the effects of N(2)O on voltage-gated Ca(2+) channels in acutely dissociated small sensory neurons of adult rat. At subanesthetic concentrations, N(2)O blocks low-voltage-activated, T-type Ca(2+) currents (T currents), but not high-voltage-activated (HVA) currents. This blockade of T currents was concentration dependent, with an IC(50) value of 45 +/- 13%, maximal block of 38 +/- 12%, and Hill coefficient of 2.6 +/- 1.0. No desensitization of the response or change in current kinetics was observed during N(2)O application. The magnitude of T current blockade by N(2)O does not seem to reflect any use- or voltage-dependent properties. In addition, T current blockade was not altered when intracellular GTP was replaced with guanosine 5'-(gamma-thio)triphosphate or guanosine 5'-0-(2-thiodiphosphate) suggesting a lack of involvement of G-proteins in the inhibition. N(2)O selectively blocked currents arising from the Ca(v)3.2 but not Ca(v)3.1 recombinant channels stably expressed in human embryonic kidney (HEK) cells in a concentration-dependent manner with an apparent affinity and potency similar to native dorsal root ganglion currents. Analogously, the block of Ca(v)3.2 T currents exhibited little voltage- or use-dependence. These data indicate that N(2)O selectively blocks T-type but not HVA Ca(2+) currents in small sensory neurons and Ca(v)3.2 currents in HEK cells at subanesthetic concentrations. Blockade of T currents may contribute to the anesthetic and/or analgesic effects of N(2)O.

Redox modulation of T-type calcium channels in rat peripheral nociceptors.

Although T-type calcium channels were first described in sensory neurons, their function in sensory processing remains unclear. In isolated rat sensory neurons, we show that redox agents modulate T currents but not other voltage- and ligand-gated channels thought to mediate pain sensitivity. Similarly, redox agents modulate currents through Ca(v)3.2 recombinant channels. When injected into peripheral receptive fields, reducing agents, including the endogenous amino acid L-cysteine, induce thermal hyperalgesia. This hyperalgesia is blocked by the oxidizing agent 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) and the T channel antagonist mibefradil. DTNB alone and in combination with mibefradil induces thermal analgesia. Likewise, L-cysteine induces mechanical DTNB-sensitive hyperalgesia in peripheral receptive fields. These data strongly suggest a role for T channels in peripheral nociception. Redox sites on T channels in peripheral nociceptors could be important targets for agents that modify pain perception.

Ducky mouse phenotype of epilepsy and ataxia is associated with mutations in the Cacna2d2 gene and decreased calcium channel current in cerebellar Purkinje cells.

The mouse mutant ducky, a model for absence epilepsy, is characterized by spike-wave seizures and ataxia. The ducky gene was mapped previously to distal mouse chromosome 9. High-resolution genetic and physical mapping has resulted in the identification of the Cacna2d2 gene encoding the alpha2delta2 voltage-dependent calcium channel subunit. Mutations in Cacna2d2 were found to underlie the ducky phenotype in the original ducky (du) strain and in a newly identified strain (du(2J)). Both mutations are predicted to result in loss of the full-length alpha2delta2 protein. Functional analysis shows that the alpha2delta2 subunit increases the maximum conductance of the alpha1A/beta4 channel combination when coexpressed in vitro in Xenopus oocytes. The Ca(2+) channel current in acutely dissociated du/du cerebellar Purkinje cells was reduced, with no change in single-channel conductance. In contrast, no effect on Ca(2+) channel current was seen in cerebellar granule cells, results consistent with the high level of expression of the Cacna2d2 gene in Purkinje, but not granule, neurons. Our observations document the first mammalian alpha2delta mutation and complete the association of each of the major classes of voltage-dependent Ca(2+) channel subunits with a phenotype of ataxia and epilepsy in the mouse.

Molecular pharmacology of T-type Ca2+ channels.

Over the past few years increasing attention has been focused on T-type calcium channels and their possible physiological and pathophysiological roles. Efforts toward elucidating the exact role(s) of these calcium channels have been hampered by the lack of T-type specific antagonists, resulting in the subsequent use of less selective calcium channel antagonists. In addition, the activity of these blockers often varies with cell or tissue type, as well as recording conditions. This review summarizes a variety of compounds that exhibit varying degrees of blocking activity towards T-type Ca2+ channels. It is designed as an aid for researchers in need of antagonists to study the biophysical and pathological nature of T-type channels, as well as a starting point for those attempting to develop potent and selective antagonists of the channel.

Functional coupling between 'R-type' Ca2+ channels and insulin secretion in the insulinoma cell line INS-1.

Among voltage-gated Ca2+ channels the non-dihydropyridine-sensitive alpha1E subunit is functionally less well characterized than the structurally related alpha1A (omega-agatoxin-IVA sensitive, P- /Q-type) and alpha1B (omega-conotoxin-GVIA sensitive, N-type) subunits. In the rat insulinoma cell line, INS-1, a tissue-specific splice variant of alpha1E (alpha1Ee) has been characterized at the mRNA and protein levels, suggesting that INS-1 cells are a suitable model for investigating the function of alpha1Ee. In alpha1E-transfected human embryonic kidney (HEK-293) cells the alpha1E-selective peptide antagonist SNX-482 (100 nM) reduces alpha1Ed- and alpha1Ee-induced Ba2+ inward currents in the absence and presence of the auxiliary subunits beta3 and alpha2delta-2 by more than 80%. The inhibition is fast and only partially reversible. No effect of SNX-482 was detected on the recombinant T-type Ca2+ channel subunits alpha1G, alpha1H, and alpha1I showing that the toxin from the venom of Hysterocrates gigas is useful as an alpha1E-selective antagonist. After blocking known components of Ca2+ channel inward current in INS-1 cells by 2 microM (+/-)-isradipine plus 0.5 microM omega-conotoxin-MVIIC, the remaining current is reduced by 100 nM SNX-482 from -12.4 +/- 1.2 pA/pF to -7.6 +/- 0.5 pA/pF (n = 9). Furthermore, in INS-1 cells, glucose- and KCl-induced insulin release are reduced by SNX-482 in a dose-dependent manner leading to the conclusion that alpha1E, in addition to L-type and non-L-type (alpha1A-mediated) Ca2+ currents, is involved in Ca2+ dependent insulin secretion of INS-1 cells.

alpha1H T-type Ca2+ channel is the predominant subtype expressed in bovine and rat zona glomerulosa.

The low voltage-activated (T-type) Ca2+ channel has been implicated in the regulation of aldosterone secretion from the adrenal zona glomerulosa by extracellular K+ levels, angiotensin II, and ACTH. However, the identity of the specific subtype mediating this regulation has not been determined. We utilized in situ hybridization to examine the distribution of three newly cloned members of the T-type Ca2+ channel family, alpha1G, alpha1H, and alpha1I, in the rat and bovine adrenal gland. Substantial expression of only the mRNA transcript for the alpha1H-subunit was detected in the zona glomerulosa of both rat and bovine. A much weaker expression signal was detected for the alpha1H transcript in the zona fasciculata of bovine. Whole cell recordings of isolated bovine adrenal zona glomerulosa cells showed the native low voltage-activated current to be inhibited by NiCl2 with an IC50 of 6.4 +/- 0.2 microM. Because the alpha1H subtype exhibits similar NiCl2 sensitivity, we propose that the alpha1H subtype is the predominant T-type Ca2+ channel present in the adrenal zona glomerulosa.

Mg(2+) block unmasks Ca(2+)/Ba(2+) selectivity of alpha1G T-type calcium channels.

We have examined permeation by Ca(2+) and Ba(2+), and block by Mg(2+), using whole-cell recordings from alpha1G T-type calcium channels stably expressed in HEK 293 cells. Without Mg(o)(2+), inward currents were comparable with Ca(2+) and Ba(2+). Surprisingly, three other results indicate that alpha1G is actually selective for Ca(2+) over Ba(2+). 1) Mg(2+) block is approximately 7-fold more potent with Ba(2+) than with Ca(2+). With near-physiological (1 mM) Mg(o)(2+), inward currents were approximately 3-fold larger with 2 mM Ca(2+) than with 2 mM Ba(2+). The stronger competition between Ca(2+) and Mg(2+) implies that Ca(2+) binds more tightly than Ba(2+). 2) Outward currents (carried by Na(+)) are blocked more strongly by Ca(2+) than by Ba(2+). 3) The reversal potential is more positive with Ca(2+) than with Ba(2+), thus P(Ca) > P(Ba). We conclude that alpha1G can distinguish Ca(2+) from Ba(2+), despite the similar inward currents in the absence of Mg(o)(2+). Our results can be explained by a 2-site, 3-barrier model if Ca(2+) enters the pore 2-fold more easily than Ba(2+) but exits the pore at a 2-fold lower rate.

Mibefradil block of cloned T-type calcium channels.

Mibefradil is a tetralol derivative chemically distinct from other calcium channel antagonists. It is a very effective antihypertensive agent that is thought to achieve its action via a higher affinity block for low-voltage-activated (T) than for high-voltage-activated (L) calcium channels. Estimates of affinity using Ba(2+) as the charge carrier have predicted a 10- to 15-fold preference of mibefradil for T channels over L channels. However, T channel IC(50) values are reported to be approximately 1 microM, which is much higher than expected for clinical efficacy because relevant blood levels of this drug are approximately 50 nM. We compared the affinity for mibefradil of the newly cloned T channel isoforms, alpha1G, alpha1H, and alpha1I with an L channel, alpha1C. In 10 mM Ba(2+), mibefradil blocked in the micromolar range and with 12- to 13-fold greater affinity for T channels than for L channels (approximately 1 microM versus 13 microM). When 2 mM Ca(2+) was used as the charge carrier, the drug was more efficacious; the IC(50) for alpha1G shifted to 270 nM and for alpha1H shifted to 140 nM, 4.5- and 9-fold higher affinity than in 10 mM Ba. The data are consistent with the idea that mibefradil competes for its binding site on the channel with the permeant species and that Ba(2+) is a more effective competitor than Ca(2+). Raising temperature to 35 degrees C reduced affinity (IC(50) 792 nM). Reducing channel availability to half increased affinity ( approximately 70 nM). This profile of mibefradil affinity makes these channels good candidates for the physiological target of this antihypertensive agent.

Overexpression of T-type calcium channels in HEK-293 cells increases intracellular calcium without affecting cellular proliferation.

Increased expression of low voltage-activated, T-type Ca(2+) channels has been correlated with a variety of cellular events including cell proliferation and cell cycle kinetics. The recent cloning of three genes encoding T-type alpha(1) subunits, alpha(1G), alpha(1H) and alpha(1I), now allows direct assessment of their involvement in mediating cellular proliferation. By overexpressing the human alpha(1G) and alpha(1H) subunits in human embryonic kidney (HEK-293) cells, we describe here that, although T-type channels mediate increases in intracellular Ca(2+) concentrations, there is no significant change in bromodeoxyuridine incorporation and flow cytometric analysis. These results demonstrate that expressions of T-type Ca(2+) channels are not sufficient to modulate cellular proliferation of HEK-293 cells.

Anticonvulsants but not general anesthetics have differential blocking effects on different T-type current variants.

The sensitivity to anticonvulsants and anesthetics of Ca(2+) currents arising from alpha1G and alpha1H subunits was examined in stably transfected HEK293 cells. For comparison, in some cases blocking effects on dorsal root ganglion (DRG) T currents were also examined under identical ionic conditions. The anticonvulsant, phenytoin, which partially blocks DRG T current, blocked alpha1G current completely but with weaker affinity ( approximately 140 microM). Among different cells, alpha1H current exhibited either of two responses to phenytoin. In one subpopulation of cells, phenytoin produced a partial, higher affinity block (IC(50) approximately 7.2 microM, maximum block approximately 43%) similar to that in DRG neurons. In other cells, phenytoin produced complete, but lower affinity, blockade (IC(50) approximately 138 microM, maximum block approximately 89%). Another anticonvulsant, alpha-methyl-alpha-phenylsuccinimide (MPS), blocked DRG current partially, but blocked both alpha1G and alpha1H currents completely with weaker affinity ( approximately 1.7 mM). These data suggest that higher affinity blockade of T-type currents by phenytoin and MPS may require additional regulatory factors that can contribute to native T-type channels. In contrast, anesthetics blocked all T current variants similarly and completely. Block of alpha1G current by anesthetics had the following order of potency: propofol (IC(50) approximately 20.5 microM) > etomidate ( approximately 161 microM) = octanol ( approximately 160 microM) > isoflurane ( approximately 277 microM) > ketamine ( approximately 1.2 mM), comparable with results on DRG T currents. Barbiturates completly blocked alpha1G currents with potency [thiopental ( approximately 280 microM), pentobarbital ( approximately 310 microM), phenobarbital ( approximately 1.54 mM)] similar to that in DRG cells. The effects of propofol, octanol, and pentobarbital on alpha1H currents were indistinguishable from effects on alpha1G currents.