Contrasting anesthetic sensitivities of T-type Ca2+ channels of reticular thalamic neurons and recombinant Ca(v)3.3 channels

Reticular thalamocortical neurons express a slowly inactivating T-type Ca(2+) current that is quite similar to that recorded from recombinant Ca(v)3.3b (alpha1Ib) channels. These neurons also express abundant Ca(v)3.3 mRNA, suggesting that it underlies the native current. Here, we test this hypothesis by comparing the anesthetic sensitivities of recombinant Ca(v)3.3b channels stably expressed in HEK 293 cells to native T channels in reticular thalamic neurons (nRT) from brain slices of young rats. Barbiturates completely blocked both Ca(v)3.3 and nRT currents, with pentobarbital being about twice more potent in blocking Ca(v)3.3 currents. Isoflurane had about the same potency in blocking Ca(v)3.3 and nRT currents, but enflurane, etomidate, propofol, and ethanol exhibited 2-4 fold higher potency in blocking nRT vs Ca(v)3.3 currents. Nitrous oxide (N(2)O; laughing gas) blocked completely nRT currents with IC(50) of 20%, but did not significantly affect Ca(v)3.3 currents at four-fold higher concentrations. In addition, we observed that in lower concentration, N(2)O reversibly increased nRT but not Ca(v)3.3 currents. In conclusion, contrasting anesthetic sensitivities of Ca(v)3.3 and nRT T-type Ca(2+) channels strongly suggest that different molecular structures of Ca(2+) channels give rise to slowly inactivating T-type Ca(2+) currents. Furthermore, effects of volatile anesthetics and ethanol on slowly inactivating T-type Ca(2+) channel variants may contribute to the clinical effects of these agents.     

5beta-reduced neuroactive steroids are novel voltage-dependent blockers of T-type Ca2+ channels in rat sensory neurons in vitro and potent peripheral analgesics in vivo

T-type Ca(2+) channels are believed to play an important role in pain perception, and anesthetic steroids such as alphaxalone and allopregnanolone, which have a 5alpha-configuration at the steroid A, B ring fusion, are known to inhibit T-type Ca(2+) channels and cause analgesia in a thermal nociceptive model (Soc Neurosci Abstr 29:657.9, 2003). To define further the structure-activity relationships for steroid analgesia, we synthesized and examined a series of 5beta-reduced steroids for their ability to induce thermal antinociception in rats when injected locally into the peripheral receptive fields of the nociceptors and studied their effects on T-type Ca(2+) channel function in vitro. We found that most of the steroids completely blocked T-type Ca(2+) currents in vitro with IC(50) values at a holding potential of -90 mV ranging from 2.8 to 40 microM. T current blockade exhibited mild voltage-dependence, suggesting that 5beta-reduced neuroactive steroids stabilize inactive states of the channel. For the most potent steroids, we found that other voltage-gated currents were not significantly affected at concentrations that produce nearly maximal blockade of T currents. All tested compounds induced dose-dependent analgesia in thermal nociceptive testing; the most potent effect (ED(50), 30 ng/100 microl) obtained with a compound [(3beta,5beta,17beta)-3-hydroxyandrostane-17-carbonitrile] that was also the most effective blocker of T currents. Compared with previously studied 5alpha-reduced steroids, these 5beta-reduced steroids are more efficacious blockers of neuronal T-type Ca(2+) channels and are potentially useful as new experimental reagents for understanding the role of neuronal T-type Ca(2+) channels in peripheral pain pathways.     

Lysophosphatidylcholine augments Ca(v)3.2 but not Ca(v)3.1 T-type Ca(2+) channel current expressed in HEK-293 cells

Lysophosphatidylcholine (LPC) has been shown to induce electrophysiological disturbances to arrhythmogenesis. However, the effects of LPC on the low-voltage-activated T-type Ca(2+) channels in the heart are not understood yet. We found that LPC increases the T-type Ca(2+) channel current (I(Ca.T)) in neonatal rat cardiomyocytes. To further investigate the underlying modulatory mechanism of LPC on T-type Ca(2+) channels, we utilized HEK-293 cells stably expressing alpha1G and alpha1H subunits (HEK-293/alpha1G and HEK-293/alpha1H), by use of patch-clamp techniques. A low concentration of LPC (10 micromol/l) significantly increased Ca(v)3.2 I(Ca.T) (alpha1H) that were similar to those observed in neonatal rat cardiomyocytes. Activation and steady-state inactivation curves were shifted in the hyperpolarized direction by 5.1 +/- 0.2 and 4.6 +/- 0.4 mV, respectively, by application of 10 micromol/l LPC. The pretreatment of cells with a protein kinase C inhibitor (chelerythrine) attenuated the effects of LPC on I(Ca.T) (alpha1H). However, the application of LPC failed to modify Ca(v)3.1 (alpha1G) I(Ca.T) at concentrations of 10-50 micromol/l. In conclusion, these data demonstrate that extracellularly applied LPC augments Ca(v)3.2 I(Ca.T) (alpha1H) but not Ca(v)3.1 I(Ca.T) (alpha1G) in a heterologous expression system, possibly by modulating protein kinase C signaling.     

Contrasting the effects of nifedipine on subtypes of endogenous and recombinant T-type Ca2+ channels

There is evidence that nifedipine (Nif) - a dihydropyridine (DHP) Ca(2+)-channel antagonist mostly known for its L-type-specific action--is capable of blocking low voltage-activated (LVA or T-type) Ca(2+) channels as well. However, the discrimination by Nif of either various endogenous T-channel subtypes, evident from functional studies, or cloned Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 T-channel alpha 1 subunits have not been determined. Here, we investigated the effects of Nif on currents induced by Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 expression in Xenopus oocytes or HEK-293 cells (I(alpha 1G), I(alpha 1H) and I(alpha 1I), respectively) and two kinetically distinct, "fast" and "slow", LVA currents in thalamic neurons (I(LVA,f) and I(LVA,s)). At voltages of the maximums of respective currents the drug most potently blocked I(alpha 1H) (IC(50)=5 microM, max block 41%) followed by I(alpha 1G) (IC(50)=109 microM, 23%) and I(alpha 1I) (IC(50)=243 microM, 47%). The mechanism of blockade included interaction with Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 open and inactivated states. Nif blocked thalamic I(LVA,f) and I(LVA,s) with nearly equal potency (IC(50)=22 microM and 28 microM, respectively), but with different maximal inhibition (81% and 51%, respectively). We conclude that Ca(v)3.2 is the most sensitive to Nif, and that quantitative characteristics of drug action on T-type Ca(2+) channels depend on cellular system they are expressed in. Some common features in the voltage- and state-dependence of Nif action on endogenous and recombinant currents together with previous data on T-channel alpha 1 subunits mRNA expression patterns in the thalamus point to Ca(v)3.1 and Ca(v)3.3 as the major contributors to thalamic I(LVA,f) and I(LVA,s), respectively.     

Short- and long-term amiodarone treatments regulate Cav3.2 low-voltage-activated T-type Ca2+ channel through distinct mechanisms

Low-voltage-activated T-type Ca2+ channels have been recognized recently in the mechanisms underlying atrial arrhythmias. However, the pharmacological effects of amiodarone on the T-type Ca2+ channel remain unclear. We investigated short- and long-term effects of amiodarone on the T-type (Cav 3.2) Ca2+ channel. The Cav3.2 alpha1H subunit derived from human heart was stably transfected into cells [human embryonic kidney (HEK)-Cav3.2] cultured with or without 5 muM amiodarone. Patch-clamp recordings in the conventional whole-cell configuration were used to evaluate the actions of amiodarone on the T-type Ca2+ channel current (ICa.T). Amiodarone blockade of ICa.T occurred in a dose- and holding potential-dependent manner, shifting the activation and the steady-state inactivation curves in the hyperpolarization direction, when amiodarone was applied immediately to the bath solution. However, when the HEK-Cav3.2 cells were incubated with 5 microM amiodarone for 72 h, ICa.T density was significantly decreased by 31.7+/-2.3% for control,-93.1+/-4.3 pA/pF (n=8), versus amiodarone,-56.5+/-3.2 pA/pF (n=13), P<0.001. After the prolonged administration of amiodarone, the activation and the steady-state inactivation curves were shifted in the depolarization direction by -7.1 (n=41) and -5.5 mV (n=37), respectively, and current inactivation was significantly delayed [time constant (tau): control, 13.3+/-1.1 ms (n=6) versus amiodarone, 39.6+/-5.5 ms (n=6) at -30 mV, P<0.001)]. Nevertheless, short-term inhibitory effects of amiodarone on the modified T-type Cav3.2 Ca2+ channel created by long-term amiodarone treatment were functionally maintained. We conclude that amiodarone exerts its short- and long-term inhibitory actions on ICa.T via distinct blocking mechanisms.     

A selective T-type Ca2+ channel blocker R(−) efonidipine

Recently, novel compound R(-) efonidipine was reported to selectively block low-voltage-activated (LVA or T-type) Ca(2+) channels in peripheral organs. We examined how R(-) efonidipine acts on T-type and high-voltage-activated (HVA) Ca(2+) channels in mammalian central nervous system (CNS) neurons. Furthermore, we compared the effects of R(-) efonidipine with those of flunarizine and mibefradil on both T-type and HVA Ca(2+) channels in rat hippocampal CA1 neurons by using the nystatin perforated-patch clamp technique. Flunarizine and mibefradil nonselectively inhibited both T-type and HVA Ca(2+) channels, though the dose-dependent blocking potency of flunarizine on T-type Ca(2+) channels was slightly stronger than that of mibefradil. In contrast, R(-) efonidipine inhibited only T-type Ca(2+) channels and did not show any effect on HVA Ca(2+) channels. The inhibitory actions of R(-) efonidipine or flunarizine were similar on both Ba(2+) and Ca(2+) current components passing through T-type Ca(2+) channels. In addition, flunarizine but not R(-) efonidipine inhibited voltage-dependent Na(+) channels and Ca(2+)-activated K(+) channels. Thus, it appears that R(-) efonidipine is a selective blocker for T-type Ca(2+) channels. It could be used as a pharmacological tool in future studies on T-type Ca(2+) channels.     

Towards selective antagonists of T-type calcium channels: design, characterization and potential applications of NNC 55-0396

NNC 55-0396 is a structural analog of mibefradil (Ro 40-5967) that inhibits both T-type and high-voltage-activated (HVA) Ca2+ channels with a higher selectivity for T-type Ca2+ channels. The inhibitory effect of mibefradil on HVA Ca2+ channels can be attributed to a hydrolyzed metabolite of the drug: the methoxy acetate side chain of mibefradil is removed by intracellular enzymes, thus it forms (1S,2S)-2-(2-(N-[(3-benzoimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl hydroxy dihydrochloride (dm-mibefradil), which causes potent inhibition of HVA Ca2+ currents. By replacing the methoxy acetate chain of mibefradil with cyclopropanecarboxylate, a more stable analog was developed (NNC 55-0396). The acute IC50 of NNC 55-0396 to block recombinant Cav3.1 T-type channels expressed in HEK293 cells is approximately 7 muM, whereas 100 microM NNC 55-0396 has no detectable effect on high voltage-activated currents in INS-1 cells. Block of T-type Ca2+ current was partially reduced by membrane hyperpolarization and was enhanced at high stimulus frequency. Washing NNC 55-0396 out of the recording chamber did not reverse the T-type Ca2+ current activity, suggesting that the compound dissolves in or passes through the plasma membrane to exert its effect; however, intracellular perfusion of the compound did not block T-type Ca2+ currents, arguing against a cytoplasmic route of action. We conclude that NNC 55-0396, by virtue of its modified structure, does not produce the metabolite that causes inhibition of L-type Ca2+ channel channels, thus rendering it more selective to T-type Ca2+ channels.     

Inhibition of recombinant low-voltage-activated Ca(2+) channels by the neuroprotective agent BW619C89 (Sipatrigine)

T-type Ca(2+) currents were recorded in 2 mM Ca(2+) from HEK 293 cells stably expressing recombinant low-voltage-activated Ca(2+) channel subunits. Current-voltage relationships revealed that these currents were low-voltage activated in nature and could be reversibly antagonised by mibefradil, a known T-type channel blocker. At a test potential of -25 mV alpha(1I)-mediated Ca(2+) currents were rapidly and reversibly inhibited by 1-100 microM BW619C89 (IC(50)=14 microM, Hill coefficient 1.3). In contrast to its actions on N-type Ca(2+) channels, a near IC(50) dose (10 microM) of BW619C89 produced no alterations in either the kinetics or voltage-dependence of T-type currents. In additional single dose experiments, currents mediated by rat alpha(1G), human alpha(1H) or human alpha(1I) channel subunits were also inhibited by BW619C89. Overall our data indicate that T-type Ca(2+) channels are more potently blocked by BW619C89 than either type-II Na(+) channels or N-type Ca(2+) channels. It seems, therefore, that inhibition of low-voltage-activated Ca(2+) channels is likely to contribute to the anticonvulsant and neuroprotective actions of this and related compounds.     

Pharmacological properties of Cav3.2, a low voltage-activated Ca2+ channel cloned from human heart

Three genes encoding T-type Ca2+ channels have been described but their correspondence to the various native T-type Ca2+ currents remains uncertain. In particular, Ca(V)3.2 (or alpha1H) was cloned from a human heart library, its message was found abundantly in cardiac tissue, and expressed Ca(V)3.2 was shown to conduct low voltage-activated currents, which inactivate rapidly and are sensitive to Ni2+ and mibefradil. These observations suggested that Ca(V)3.2 might encode native cardiac T-type Ca2+ channels but more information on the pharmacology of Ca(V)3.2 was needed to confirm this hypothesis. In the present study, we compare the pharmacology of Ca(V)3.2 expressed in HEK293 cells and of native T-type Ca2+ channels in guinea pig atrial myocytes ("native-T"). (1) Ca(V)3.2 and native-T are insensitive to TTX and to toxins selective for N-, P-, or Q-type Ca2+ channels (omega-CTx-GVIA, omega-Aga-IVA, omega-CTx-MVIIC). (2) The half-maximal blocking concentration (IC50) of mibefradil on Ca(V)3.2 is near that on native-T and the block is similarly voltage-dependent. (3) Ca(V)3.2 is five- to sixfold less sensitive than native-T to the 1,4-dihydropyridine (DHP) amlodipine, suggesting a difference in the DHP binding site. (4) Both channels display similar (but not identical) sensitivities to the inorganic blockers Ni2+ and Cd2+ and the IC50s are in the range of values found for T-type Ca2+ currents in other cell types. (5) Ni2+ shifts the voltage dependence of Ca(V)3.2 activation but not that of native-T. The many similarities between the two channels support the contention that Ca(V)3.2 encodes cardiac T-type Ca2+ channels. The slight differences may be due to species variations and/or to the choice of splice variant.     

Potent inhibition of a recombinant low voltage-activated Ca(2+) channel by SB-209712

T-type Ca(2+) currents were recorded in 2 mM Ca(2+) from HEK293 cells stably expressing the low voltage-activated Ca(2+) channel sub-unit alpha(1I). These currents were inhibited by the known Ca(2+) channel antagonist mibefradil with an IC(50) close to 1 microM. SB-209712 (1,6,bis?1-[4-(3-phenylpropyl)piperidinyl]?hexane), a compound originally developed as a high voltage-activated Ca(2+) channel blocker, proved to be a more potent T-type channel antagonist, exhibiting an IC(50) in the region of 500 nM. The antagonism produced by SB-209712 was reversed following drug removal and the observed antagonism exhibited little or no voltage-dependence with respect to either holding or test potential. These data indicate that SB-209712 is amongst the most potent known non-peptide T-type channel antagonists and thus may have some use in understanding the role of these channels in cellular function.     

Validation of high throughput screening assays against three subtypes of Ca(v)3 T-type channels using molecular and pharmacologic approaches

T-type Ca(2+) channels encoded by voltage-gated Ca(2+) channel (Ca(v)) 3.1, 3.2, and 3.3 genes play important physiological roles and serve as therapeutic targets for neurological and cardiovascular disorders. Currently there is no selective T-channel blocker. To screen for such a blocker, we developed three stable cell lines expressing human recombinant Ca(v)3.1, 3.2, or 3.3 channels and then examined their usefulness in high throughput screens. All three cell lines displayed an increase in intracellular Ca(2+) in response to changes in extracellular Ca(2+) as detected with Ca(2+)-sensitive dyes using a fluorometric imaging plate reader (FLIPR [Molecular Devices, Sunnyvale, CA] or FlexStation [Molecular Devices]). The signal-to-noise ratio was 2-4. Co-expression of Ca(v)3.2 with a mouse leak K(+) channel, which by virtue of being open at rest hyperpolarizes the cell membrane, blocked the fluorescent signal. Co-addition of KCl to these cells induced a Ca(2+) signal that was similar to that observed in the cell line expressing Ca(v)3.2 alone. These results confirm that the detection of intracellular Ca(2+) increase in cells expressing Ca(v)3.2 alone results from Ca(2+) entry through channels that are open at the resting membrane potential of each cell line (i.e., window currents). Testing known drugs on Ca(v)3 channels showed that block could be reliably detected using the FlexStation assay, FLIPR assay, or voltage clamp recordings using the IonWorks HT system (Molecular Devices). These results support the use of the FLIPR window current assay for primary drug screening and high throughput patch recordings for secondary screening of novel T-channel blockers.     

Involvement of the endogenous hydrogen sulfide/Ca(v) 3.2 T-type Ca(2+) channel pathway in cystitis-related bladder pain in mice

BACKGROUND AND PURPOSE Hydrogen sulfide (H(2) S), generated by enzymes such as cystathionine-γ-lyase (CSE) from L-cysteine, facilitates pain signals by activating the Ca(v) 3.2 T-type Ca(2+) channels. Here, we assessed the involvement of the CSE/H(2) S/Ca(v) 3.2 pathway in cystitis-related bladder pain. EXPERIMENTAL APPROACH Cystitis was induced by i.p. administration of cyclophosphamide in mice. Bladder pain-like nociceptive behaviour was observed and referred hyperalgesia was evaluated using von Frey filaments. Phosphorylation of ERK in the spinal dorsal horn was determined immunohistochemically following intravesical administration of NaHS, an H(2) S donor. KEY RESULTS Cyclophosphamide caused cystitis-related symptoms including increased bladder weight, accompanied by nociceptive changes (bladder pain-like nociceptive behaviour and referred hyperalgesia). Pretreatment with DL-propargylglycine, an inhibitor of CSE, abolished the nociceptive changes and partly prevented the increased bladder weight. CSE protein in the bladder was markedly up-regulated during development of cystitis. Mibefradil or NNC 55-0396, blockers of T-type Ca(2+) channels, administered after the symptoms of cystitis appeared, reversed the nociceptive changes. Further, silencing of Ca(v) 3.2 protein by repeated intrathecal administration of mouse Ca(v) 3.2-targeting antisense oligodeoxynucleotides also significantly attenuated the nociceptive changes, but not the increased bladder weight. Finally, the number of cells staining positive for phospho-ERK was increased in the superficial layer of the L6 spinal cord after intravesical administration of NaHS, an effect inhibited by NNC 55-0396. CONCLUSION AND IMPLICATIONS Endogenous H(2) S, generated by up-regulated CSE, caused bladder pain and referred hyperalgesia through the activation of Ca(v) 3.2 channels, one of the T-type Ca(2+) channels, in mice with cyclophosphamide-induced cystitis.     

Voltage-dependent and frequency-independent inhibition of recombinant Cav3.2 T-type Ca2+ channel by bepridil

Effects of bepridil on the low voltage-activated T-type Ca2+ channel (CaV3.2) current stably expressed in human embryonic kidney (HEK)-293 cells were examined using patch-clamp techniques. Bepridil potently inhibited ICa,T with a markedly voltage-dependent manner; the IC50 of bepridil was 0.4 micromol/l at the holding potential of -70 mV, which was 26 times as potent as that at -100 mV (10.6 micromol/l). Steady-state inactivation curve (8.4 +/- 1.7 mV) and conductance curve (5.9 +/- 1.9 mV) were shifted to the hyperpolarized potential by 10 micromol/l bepridil. Bepridil exerted the tonic blocking action but not the use-dependent block. Bepridil had no effect on the recovery from inactivation of T-type Ca2+ channels. Thus, high efficacy of bepridil for terminating atrial fibrillation and atrial flutter may be considered to be attributed, at least in a part, to the T-type Ca2+ channel-blocking actions.     

Inhibition of neuronal high-voltage activated calcium channels by the omega-phoneutria nigriventer Tx3-3 peptide toxin

We have investigated the effect of omega-PnTx3-3 (referred to in previous papers simply as Tx3-3), a peptide toxin from the venom of the spider Phoneutria nigriventer, on neuronal high-voltage activated (HVA) Ca(2+) channels, using whole-cell patch-clamp. omega-PnTx3-3 (120 nM) blocked 74+/-8% of the total HVA Ca(2+) currents of cerebellar granule neurones, without affecting the low-voltage activated (LVA) current. P/Q/R-type currents in cerebellar granule neurones, isolated using 4 microM nicardipine and 100 nM omega-conotoxin GVIA, were markedly (79+/-6%) inhibited by 60 nM omega-PnTx3-3. R-type currents, isolated either by additional application of 0.5-1 microM of omega-agatoxin IVA or by pre-incubation with 5 microM omega-conotoxin MVIIC were inhibited almost totally by 120 nM of omega-PnTx3-3. omega-PnTx3-3 reversibly altered the kinetics of the P/Q/R current, increasing the degree of inactivation that occurred during a 50 ms pulse from 20% to 40%. N-type currents, recorded from neuroblastoma N18 cells, were partially (34+/-2%) inhibited by 320 nM omega-PnTx3-3. L-type currents, recorded from GH3 cells, were partially (45+/-12%) inhibited by 80 nM omega-PnTx3-3. We conclude that omega-PnTx3-3 inhibits all known HVA Ca(2+) channels, and most effectively the P/Q- and R-type currents.     

Colonic hydrogen sulfide-induced visceral pain and referred hyperalgesia involve activation of both Ca(v)3.2 and TRPA1 channels in mice

Luminal hydrogen sulfide (H(2)S), a gasotransmitter, causes colonic pain / referred hyperalgesia in mice, most probably via activation of T-type Ca(2+) channels. Here we analyzed the mechanisms for H(2)S-induced facilitation of colonic pain signals. Intracolonic administration of NaHS, an H(2)S donor, evoked visceral pain-like nociceptive behavior and referred hyperalgesia in mice, an effect abolished by NNC 55-0396, a selective T-type Ca(2+)-channel blocker, or by knockdown of Ca(v)3.2. AP18, a TRPA1 blocker, also prevented the NaHS-induced colonic pain and referred hyperalgesia. These findings demonstrate that H(2)S-induced colonic pain and referred hyperalgesia require activation of both Ca(v)3.2 and TRPA1 channels in mice.     

Blockade of Ba2+ current through human alpha1E channels by two steroid analogs, (+)-ACN and (+)-ECN.

Previous work suggests that different neuroactive steroids may exhibit some selectivity in their blocking effects on different high-voltage activated (HVA) Ca2+ currents. At least some of these effects appear to involve direct blocking actions on Ca2+ channels. Thus, direct investigation of the effects of various steroids on cloned Ca2+ channel variants may lead to the development of potent and selective small-molecular weight Ca2+ channel blockers. Here we examine the effects of two steroids on a cloned human alpha1E Ca2+ channel both with and without a beta3 subunit, when expressed in HEK293 cells. One compound, (+)-ACN, has been previously shown to block N-, Q-, and R-subtypes of HVA current without affecting L- and P-type current. The second compound, (+)-ECN, weakly blocks total HVA current in hippocampal neurons. (+)-ECN differs from (+)-ACN in lacking effects on GABA receptors, but shares with (+)-ACN an ability to partially inhibit T current in DRG neurons (Todorovic, S.M., Prakriya, M., Nakashima, Y.M. et al., 1998. Enantioselective blockade of T-type Ca2+ current in adult rat sensory neurons by a steroid lacking GABA-mimetic activity. Mol. Pharmacol. 54, 918-927). (+)-ACN can block 100% of Ba2+ current in HEK cells arising either from the alpha1E subunit (IC50 approximate to 10 microM) or the alpha1Ebeta3 combination (IC50 approximate to 5 microM), while (+)-ECN maximally blocks only about 80% of the alpha1E (10 microM) or alpha1Ebeta3 (16 microM) current. Blockade by (+)-ACN exhibits several differences from blockade by (+)-ECN. (+)-ACN increases the apparent rate of onset of inactivation, particularly for the alpha1E variant, slows recovery from inactivation, and more profoundly shifts the voltage-dependence of current availability for both alpha1E and alpha1Ebeta3 variants than does (+)-ECN. Although the complexity of the normal inactivation kinetics of alpha1E variants makes interpretation of the (+)-ACN-induced kinetic alterations difficult, the results suggest that the two steroids are to some extent acting by distinct mechanisms, and perhaps at different sites.     

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.     

Ethanol withdrawal seizure susceptibility is associated with upregulation of L- and P-type Ca2+ channel currents in rat inferior colliculus neurons

Calcium currents in the inferior colliculus (IC) are thought to play an important role in ethanol withdrawal hyperexcitability. Here, we report on the modulation of Ca(2+) channel currents in acutely dissociated IC neurons of rats, exhibiting higher incidence of audiogenic seizures when subjected to ethanol withdrawal. Whole cell Ca(2+) channel currents were activated by depolarizing pulses from a holding potential of -90 mV, in 10 mV increments, using barium (Ba(2+)) as the charge carrier. The high threshold voltage-activated (HVA) Ca(2+) channel current density increased significantly in IC neurons following ethanol withdrawal. The gating parameters of HVA Ca(2+) channel currents were only slightly altered, while the fraction of current that did not fully inactivate at positive potentials increased significantly following ethanol withdrawal. Pharmacological dissection of HVA Ca(2+) channel currents suggested that the enhanced current, associated with increased incidence of audiogenic seizures following ethanol withdrawal, was carried by L- and P-type Ca(2+) channels. The upregulation of L- and P-type currents may be responsible for IC neuronal hyperexcitability associated with increased susceptibility to ethanol withdrawal seizures.     

Effect of cilnidipine on L- and T-type calcium currents in guinea pig ventricle and action potential in rabbit sinoatrial node

Cilnidipine, a dihydropyridine Ca(2+) channel antagonist, is known to have inhibitory effects on both L- and N-type Ca(2+) currents. In the present study, we examined the effect of cilnidipine on myocardial L- and T-type Ca(2+) currents and sinoatrial node action potential configuration. In voltage clamped guinea pig ventricular myocytes, cilnidipine concentration-dependently decreased L- and T-type Ca(2+) currents. In rabbit sinoatrial node tissue, cilnidipine increased cycle length through reduction of phase 4 depolarization slope. In conclusion, cilnidipine has inhibitory effects on T-type Ca(2+) current, which may contribute to its negative chronotropic potency.