Attenuated pain responses in mice lacking Ca(V)3.2 T-type channels

Although T-type Ca(2+) channels are implicated in nociception, the function of specific subtypes has not been well defined. Here, we compared pain susceptibility in mice lacking Ca(V)3.2 subtype of T-type Ca(2+) channels (Ca(V)3.2(-/-)) with wild-type littermates in various behavioral models of pain to explore the roles of Ca(V)3.2 in the processing of noxious stimuli in vivo. In acute mechanical, thermal and chemical pain tests, Ca(V)3.2(-/-) mice showed decreased pain responses compared to wild-type mice. Ca(V)3.2(-/-) mice also displayed attenuated pain responses to tonic noxious stimuli such as intraperitoneal injections of irritant agents and intradermal injections of formalin. In spinal nerve ligation-induced neuropathic pain, however, behavioral responses of Ca(V)3.2(-/-) mice were not different from those of wild-type mice. The present study reveals that the Ca(V)3.2 subtype of T-type Ca(2+) channels are important in the peripheral processing of noxious signals, regardless of modality, duration or affected tissue type.     

Expression and Regulation of Cav3.2 T-Type Calcium Channels during Inflammatory Hyperalgesia in Mouse Dorsal Root Ganglion Neurons

The Cav3.2 isoform of the T-type calcium channel is expressed in primary sensory neurons of the dorsal root ganglion (DRG), and these channels contribute to nociceptive and neuropathic pain in rats. However, there are conflicting reports on the roles of these channels in pain processing in rats and mice. In addition, the function of T-type channels in persistent inflammatory hyperalgesia is poorly understood. We performed behavioral and comprehensive histochemical analyses to characterize Cav3.2-expressing DRG neurons and examined the regulation of T-type channels in DRGs from C57BL/6 mice with carrageenan-induced inflammatory hyperalgesia. We show that approximately 20% of mouse DRG neurons express Cav3.2 mRNA and protein. The size of the majority of Cav3.2-positive DRG neurons (69 ± 8%) ranged from 300 to 700 μm2 in cross-sectional area and 20 to 30 μm in estimated diameter. These channels co-localized with either neurofilament-H (NF-H) or peripherin. The peripherin-positive cells also overlapped with neurons that were positive for isolectin B4 (IB4) and calcitonin gene-related peptide (CGRP) but were distinct from transient receptor potential vanilloid 1 (TRPV1)-positive neurons during normal mouse states. In mice with carrageenan-induced inflammatory hyperalgesia, Cav3.2 channels, but not Cav3.1 or Cav3.3 channels, were upregulated in ipsilateral DRG neurons during the sub-acute phase. The increased Cav3.2 expression partially resulted from an increased number of Cav3.2-immunoreactive neurons; this increase in number was particularly significant for TRPV1-positive neurons. Finally, preceding and periodic intraplantar treatment with the T-type calcium channel blockers mibefradil and NNC 55-0396 markedly reduced and reversed mechanical hyperalgesia during the acute and sub-acute phases, respectively, in mice. These data suggest that Cav3.2 T-type channels participate in the development of inflammatory hyperalgesia, and this channel might play an even greater role in the sub-acute phase of inflammatory pain due to increased co-localization with TRPV1 receptors compared with that in the normal state.     

The biochemical activation of T-type Ca2+ channels in HEK293 cells stably expressing alpha1G and Kir2.1 subunits

In order to investigate the currently unknown cellular signaling pathways of T-type Ca(2+) channels, we decided to construct a new cell line which would stably express alpha(1G) and Kir2.1 subunits in HEK293 cells (HEK293/alpha(1G)/Kir2.1). Compared to cells which only expressed alpha(1G) (HEK293/alpha(1G)), HEK293/alpha(1G)/Kir2.1 cells produced an enormous inward rectifying current which was blocked by external Ba(2+) and Cs(+) in a concentration-dependent manner. The expression of Kir2.1 channels contributed significantly to the shift of membrane potential from -12.2+/-2.8 to -57.3+/-3.7mV. However, biophysical and pharmacological properties of alpha(1G)-mediated Ca(2+) channels remained unaffected by the expression of Kir2.1 subunits, except for the enlarging of the window current region. Biochemical activation of alpha(1G) channels using 150mM KCl brought about an increase in [Ca(2+)](i), which was blocked by mibefradil, the T-type Ca(2+) channel blocker. These data suggest that the HEK293/alpha(1G)/Kir2.1 cell line would have potential uses in the study of T-type Ca(2)(+) channel-mediated signaling pathways and possibly useful in the development of new therapeutic drugs associated with T-type Ca(2)(+) channels.     

Lack of the burst firing of thalamocortical relay neurons and resistance to absence seizures in mice lacking alpha(1G) T-type Ca(2+) channels

T-type Ca(2+) currents have been proposed to be involved in the genesis of spike-and-wave discharges, a sign of absence seizures, but direct evidence in vivo to support this hypothesis has been lacking. To address this question, we generated a null mutation of the alpha(1G) subunit of T-type Ca(2+) channels. The thalamocortical relay neurons of the alpha(1G)-deficient mice lacked the burst mode firing of action potentials, whereas they showed the normal pattern of tonic mode firing. The alpha(1G)-deficient thalamus was specifically resistant to the generation of spike-and-wave discharges in response to GABA(B) receptor activation. Thus, the modulation of the intrinsic firing pattern mediated by alpha(1G) T-type Ca(2+) channels plays a critical role in the genesis of absence seizures in the thalamocortical pathway.     

Structure and function of neuronal Ca2+ channels and their role in neurotransmitter release

Electrophysiological studies of neurons reveal different Ca2+ currents designated L-, N-, P-, Q-, R-, and T-type. High-voltage-activated neuronal Ca2+ channels are complexes of a pore-forming alpha 1 subunit of about 190-250 kDa, a transmembrane, disulfide-linked complex of alpha 2 and delta subunits, and an intracellular beta subunit, similar to the alpha 1, alpha 2 delta, and beta subunits previously described for skeletal muscle Ca2+ channels. The primary structures of these subunits have all been determined by homology cDNA cloning using the corresponding subunits of skeletal muscle Ca2+ channels as probes. In most neurons, L-type channels contain alpha 1C or alpha 1D subunits, N-type contain alpha 1B subunits, P- and Q-types contain alternatively spliced forms of alpha 1A subunits, R-type contain alpha 1E subunits, and T-type contain alpha 1G or alpha 1H subunits. Association with different beta subunits also influences Ca2+ channel gating substantially, yielding a remarkable diversity of functionally distinct molecular species of Ca2+ channels in neurons.     

Conducted vasoconstriction in rat mesenteric arterioles: role for dihydropyridine-insensitive Ca(2+) channels

The aim of this study was to evaluate the role of voltage-operated Ca(2+) channels in the initiation and conduction of vasoconstrictor responses to local micropipette electrical stimulation of rat mesenteric arterioles (28 +/- 1 microm, n = 79) in vivo. Local and conducted (600 microm upstream from the pipette) vasoconstriction was not blocked by TTX (1 micromol/l, n = 5), nifedipine, or nimodipine (10 micromol/l, n = 9). Increasing the K(+) concentration of the superfusate to 75 mmol/l did not evoke vasoconstriction, but this depolarizing stimulus reversibly abolished vasoconstrictor responses to current stimulation (n = 7). Addition of the T-type Ca(2+) antagonist mibefradil (10 micromol/l, n = 6) to the superfusate reversibly blocked local and conducted vasoconstriction to current stimulation. With the use of RT-PCR techniques, it was demonstrated that rat mesenteric arterioles <40 microm do not express mRNA for L-type Ca(2+) channels (alpha(1C)-subunit), whereas mRNA coding for T-type subunits was found (alpha(1G)- and alpha(1H)-subunits). The data indicate that L-type Ca(2+) channels are absent from rat mesenteric arterioles (<40 microm). Rather, the vasoconstrictor responses appear to rely on other types of voltage-gated, dihydropyridine-insensitive Ca(2+) channels, possibly of the T-type.     

Remodeling excitation-contraction coupling of hypertrophied ventricular myocytes is dependent on T-type calcium channels expression

We utilized Wistar rats with monocrotaline (MCT)-induced right ventricular hypertrophy (RVH) in order to evaluate the T-type Ca2+ channel current (ICaT) for myocardial contraction. RT-PCR provides that mRNA for T-type Ca2+ channel alpha1-subunits in hypertrophied myocytes was significantly higher than those in control rats (alpha1G; 264+/-36%, alpha1H; 191+/-34%; P<0.05). By whole-cell patch-clamp study, ICaT was recorded only in hypertrophied myocytes but not in control myocytes. The application of 50 nmol/L nifedipine reduced the twitch tension of the right ventricles equally in the control and RVH rats. On the other hand, 0.5 micromol/L mibefradil, a T-type Ca2+ channel blocker, strongly inhibited the twitch tension of the RVH muscle (control 6.4+/-0.8% vs. RVH 20.0+/-2.3% at 5 Hz; P<0.01). In conclusion, our results indicate the functional expression of T-type Ca2+ channels in the hypertrophied heart and their contribution to the remodeling of excitation-contraction coupling in the cardiac myocyte.     

Regulation of T-type calcium channels in the peripheral pain pathway

Recent evidence strongly suggests that both central and peripheral T-type Ca(2+) channels enhance somatic and visceral nociceptive inputs, as well as that regulation of T-type Ca(2+) channel function can result in significant changes of pain threshold in a variety of animal models. Therefore, T-type Ca(2+) channels in peripheral and central pain pathways, although previously unrecognized, may have great importance as targets for developing new therapies against pain. This is particularly critical in cases in which currently available treatments are limited due to serious side effects or are not consistently effective (e.g., chronic neuropathic pain). In this review, we summarize recent studies of the regulation of T-type channels in peripheral sensory neurons by means of redox agents and neuroactive steroids, as well as studies of the function of these channels in the pathophysiology of neuropathic pain.     

Atorvastatin inhibits angiotensin II-induced T-type Ca2+ channel expression in endothelial cells

Ca2+ channels are involved in the regulation of vascular functions. Angiotensin II is implicated in the development of atherosclerosis and vascular remodeling. In this study, we demonstrated that angiotensin II preferentially increased the expression of alpha1G, a T-type Ca2+ channel subunit, via AT1 receptors in endothelial cells. Angiotensin II-induced expression of alpha1G was inhibited by pretreatment with atorvastatin and the MEK1/2 inhibitor, PD98059. The effect of atorvastatin was reversed by mevalonate and farnesyl pyrophosphate which implicates the activation of the small GTP-binding protein, Ras. Our data indicate that angiotensin II induces alpha1G expression in endothelial cells via AT1 receptors, Ras and MEK. Angiotensin II-induced migration of endothelial cells in a wound healing model was inhibited by incubation with mibefradil, a T-type Ca2+ channel blocker. Our data indicate that angiotensin II induces T-type Ca2+ channels in endothelial cells, which may play a role in the development of vascular disorders.     

T-type calcium channels and tumor proliferation

The role of T-type Ca2+ channels in proliferation of tumor cells is reviewed. Intracellular Ca2+ is important in controlling proliferation as evidenced by pulses, or oscillations, of intracellular Ca2+ which occur in a cell cycle-dependent manner in many tumor cells. Voltage-gated calcium channels, such as the T-type Ca2+ channel, are well suited to participate in such oscillations due to their unique activation/inactivation properties. Expression of the T-type Ca2+ channels has been reported in numerous types of tumors, and has been shown to be cell cycle-dependent. Overexpression of the alpha1 subunit of T-type Ca2+ channels in human astrocytoma, neuroblastoma and renal tumor cell lines enhanced proliferation of these cells. In contrast, targeting of the alpha1 subunit of the T-type calcium channel via siRNA decreased proliferation of these cells. A Ca2+ oscillatory model is proposed involving potassium channels, Ca2+ stores and Ca2+ exchangers/transporters. A review of T-type channel blockers is presented, with a focus on mibefradil-induced inhibition of proliferation. The development of newer blockers with higher selectivity and less potential side effects are discussed. The conclusion reached is that calcium channel blockers serve as a potential therapeutic approach for tumors whose proliferation depends on T-type calcium channel expression.     

Nickel block of three cloned T-type calcium channels: low concentrations selectively block alpha1H

Nickel has been proposed to be a selective blocker of low-voltage-activated, T-type calcium channels. However, studies on cloned high-voltage-activated Ca(2+) channels indicated that some subtypes, such as alpha1E, are also blocked by low micromolar concentrations of NiCl(2). There are considerable differences in the sensitivity to Ni(2+) among native T-type currents, leading to the hypothesis that there may be more than one T-type channel. We confirmed part of this hypothesis by cloning three novel Ca(2+) channels, alpha1G, H, and I, whose currents are nearly identical to the biophysical properties of native T-type channels. In this study we examined the nickel block of these cloned T-type channels expressed in both Xenopus oocytes and HEK-293 cells (10 mM Ba(2+)). Only alpha1H currents were sensitive to low micromolar concentrations (IC(50) = 13 microM). Much higher concentrations were required to half-block alpha1I (216 microM) and alpha1G currents (250 microM). Nickel block varied with the test potential, with less block at potentials above -30 mV. Outward currents through the T channels were blocked even less. We show that depolarizations can unblock the channel and that this can occur in the absence of permeating ions. We conclude that Ni(2+) is only a selective blocker of alpha1H currents and that the concentrations required to block alpha1G and alpha1I will also affect high-voltage-activated calcium currents.     

Molecular and functional properties of the human alpha(1G) subunit that forms T-type calcium channels

We describe here several novel properties of the human alpha(1G) subunit that forms T-type calcium channels. The partial intron/exon structure of the corresponding gene CACNA1G was defined and several alpha(1G) isoforms were identified, especially two isoforms that exhibit a distinct III-IV loop: alpha(1G-a) and alpha(1G-b). Northern blot and dot blot analyses indicated that alpha(1G) mRNA is predominantly expressed in the brain, especially in thalamus, cerebellum, and substantia nigra. Additional experiments have also provided evidence that alpha(1G) mRNA is expressed at a higher level during fetal life in nonneuronal tissues (i.e. kidney, heart, and lung). Functional expression in HEK 293 cells of a full-length cDNA encoding the shortest alpha(1G) isoform identified to date, alpha(1G-b), resulted in transient, low threshold activated Ca(2+) currents with the expected permeability ratio (I(Sr) > I(Ca) >/= I(Ba)) and channel conductance ( approximately 7 pS). These properties, together with slowly deactivating tail currents, are typical of those of native T-type Ca(2+) channels. This alpha(1G)-related current was inhibited by mibefradil (IC(50) = 2 microM) and weakly blocked by Ni(2+) ions (IC(50) = 148 microM) and amiloride (IC(50) > 1 mM). We showed that steady state activation and inactivation properties of this current can generate a "window current" in the range of -65 to -55 mV. Using neuronal action potential waveforms, we show that alpha(1G) channels produce a massive and sustained Ca(2+) influx due to their slow deactivation properties. These latter properties would account for the specificity of Ca(2+) influx via T-type channels that occurs in the range of physiological resting membrane potentials, differing considerably from the behavior of other Ca(2+) channels.     

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.     

Low voltage-activated calcium channels in vascular smooth muscle: T-type channels and AVP-stimulated calcium spiking

An important path of extracellular calcium influx in vascular smooth muscle (VSM) cells is through voltage-activated Ca2+ channels of the plasma membrane. Both high (HVA)- and low (LVA)-voltage-activated Ca2+ currents are present in VSM cells, yet little is known about the relevance of the LVA T-type channels. In this report, we provide molecular evidence for T-type Ca2+ channels in rat arterial VSM and characterize endogenous LVA Ca2+ currents in the aortic smooth muscle-derived cell line A7r5. AVP is a vasoconstrictor hormone that, at physiological concentrations, stimulates Ca2+ oscillations (spiking) in monolayer cultures of A7r5 cells. The present study investigated the role of T-type Ca2+ channels in this response with a combination of pharmacological and molecular approaches. We demonstrate that AVP-stimulated Ca2+ spiking can be abolished by mibefradil at low concentrations (<1 microM) that should not inhibit L-type currents. Infection of A7r5 cells with an adenovirus containing the Cav3.2 T-type channel resulted in robust LVA Ca2+ currents but did not alter the AVP-stimulated Ca2+ spiking response. Together these data suggest that T-type Ca2+ channels are necessary for the onset of AVP-stimulated calcium oscillations; however, LVA Ca2+ entry through these channels is not limiting for repetitive Ca2+ spiking observed in A7r5 cells.     

T-type Ca2+ channel expression in human esophageal carcinomas: a functional role in proliferation

In the present study the role of T-type Ca(2+) channels in cancer cell proliferation was examined. Seventeen human esophageal cancer cell lines were screened for T-type channels using RT-PCR and voltage-clamp recordings. mRNAs for all three T-type channel alpha(1)-subunits (alpha(1G), alpha(1H), and alpha(1I)) were detected in all 17 cell lines: either alpha(1H) alone, alpha(1H) and alpha(1G), or all three T-type alpha(1)-subunits. Eleven cell lines were further subjected to voltage-clamp recordings: one, i.e. the TE8 cell line, was found to exhibit a typical T-type current while others exhibited a minimal or no T-type current. Cell proliferation assays were performed in the presence or absence of T-type channel blocker mibefradil in KYSE150, KYSE180 and TE1 cells expressing mRNA for T-type channel alpha(1)-subunits but lacking T-type current, and TE8 cells exhibiting T-type current. Only TE8 cell proliferation was reduced by mibefradil. Silencing the alpha(1G)-gene that encodes functional T-type Ca(2+) channels in TE8 cells with type-specific shRNA transduction also significantly decreased TE8 cell proliferation. The reduction of cell proliferation in TE8 cells was found to be associated with an up-regulation of p21(CIP1). Moreover, p53 silencing nearly abolished the up-regulation of p21(CIP1) resulting from mibefradil T-type channel blockade. Together, these findings suggest a functional role of T-type channels in certain esophageal carcinomas, and that inhibition of T-type channels reduces cell proliferation via a p53-dependent p21(CIP1) pathway.     

Role of spinal voltage-dependent calcium channel alpha 2 delta-1 subunit in the expression of a neuropathic pain-like state in mice

The present study was undertaken to investigate the role of spinal voltage-dependent calcium channel alpha(2)delta-1 subunit in the expression of a neuropathic pain-like state induced by partial sciatic nerve ligation in mice. In cultured spinal neurons, gabapentin (GBP), which displays the inhibitory effect of alpha(2)delta-1 subunit, suppressed the extracellular Ca(2+) influx induced by KCl, whereas it failed to inhibit the intracellular Ca(2+) release induced by inositol-1,4,5-triphosphate. Seven days after sciatic nerve ligation, the protein level of alpha(2)delta-1 subunit in the ipsilateral spinal cord was clearly increased compared to that observed in sham-operated mice. In addition, the mRNA level of alpha(2)delta-1 subunit was significantly increased in the dorsal root ganglion, but not in the spinal cord, of nerve-ligated mice. Under these conditions, a marked decrease in the latency of paw-withdrawal against a thermal stimulation and tactile stimulation, induced by sciatic nerve ligation was abolished by repeated intrathecal (i.t.) treatment with GBP. Additionally, the persistent reduction in the nociceptive threshold by i.t. treatment with GBP at the early stage of the neuropathic pain-like state was maintained for 7 days even after GBP withdrawal. It is of interest to note that a single i.t. post-injection of GBP showed a marked and transient inhibitory effect on the developed neuropathic pain-like state, whereas repeated i.t. post-treatment with GBP produced a persistent inhibitory effect during the treatment. In conclusion, we propose here that the neuropathic pain-like state with sciatic nerve ligation is associated with the increased level of the alpha(2)delta-1 subunit of Ca(2+) channels at the sensory nerve terminal in the spinal dorsal horn of mice. Furthermore, the present data provide evidence that the neuropathic pain may be effectively controlled by repeated treatment with GBP at the early stage.     

Low-threshold exocytosis induced by cAMP-recruited CaV3.2 (alpha1H) channels in rat chromaffin cells

We have studied the functional role of CaV3 channels in triggering fast exocytosis in rat chromaffin cells (RCCs). CaV3 T-type channels were selectively recruited by chronic exposures to cAMP (3 days) via an exchange protein directly activated by cAMP (Epac)-mediated pathway. Here we show that cAMP-treated cells had increased secretory responses, which could be evoked even at very low depolarizations (-50, -40 mV). Potentiation of exocytosis in cAMP-treated cells did not occur in the presence of 50 microM Ni2+, which selectively blocks T-type currents in RCCs. This suggests that the "low-threshold exocytosis" induced by cAMP is due to increased Ca2+ influx through cAMP-recruited T-type channels, rather than to an enhanced secretion downstream of Ca2+ entry, as previously reported for short-term cAMP treatments (20 min). Newly recruited T-type channels increase the fast secretory response at low voltages without altering the size of the immediately releasable pool. They also preserve the Ca2+ dependence of exocytosis, the initial speed of vesicle depletion, and the mean quantal size of single secretory events. All this indicates that cAMP-recruited CaV3 channels enhance the secretory activity of RCCs at low voltages by coupling to the secretory apparatus with a Ca2+ efficacy similar to that of already existing high-threshold Ca2+ channels. Finally, using RT-PCRs we found that the fast inactivating low-threshold Ca2+ current component recruited by cAMP is selectively associated to the alpha1H (CaV3.2) channel isoform.     

Frequency-dependent blockade of T-type Ca2+ current by efonidipine in cardiomyocytes

Efonidipine is a dihydropyridine Ca2+ antagonist with inhibitory effects on both L-type and T-type Ca2+ channels and potent bradycardiac activity especially in patients with high heart rate. In the present study, we examined the frequency dependence of efonidipine action on the T-type Ca2+ channel in isolated guinea-pig ventricular myocytes. The potency of efonidipine to inhibit the T-type Ca2+ current was higher under higher stimulation frequencies. The IC50 values were 1.3 x 10(-8), 2.0 x 10(-6) and 6.3 x 10(-6) M under stimulation frequencies of 1, 0.2 and 0.05 Hz, respectively. The reduction of T-type Ca2+ current amplitude was not accompanied by change in the time course of current decay. Efonidipine (10 microM) inhibited T-type Ca2+ current elicited by depolarization from holding potentials ranging from -90 to -30 mV by about 30%; the voltage-dependence of steady-state inactivation was not changed by the drug. Efonidipine slowed the recovery from inactivation following an inactivating prepulse. In conclusion, efonidipine was shown to have frequency-dependent inhibitory effects on the T-type Ca2+ channel, which could be explained by slow dissociation of the drug from the inactivated state of the channel.