regulation of the calcium channel α1g subunit by divalent cations and organic blockers

Neuropharmacology 39 (2000) 1254–1266www.elsevier.com/locate/neuropharm

Regulation of the calcium channelα1G subunit by divalent cationsand organic blockers

L. Lacinova1,*, N. Klugbauer, F. HofmannInstitut fur Pharmakologie und Toxikologie der Technischen Universita¨t Munchen, Biedersteiner Str. 29, 80802 Mu¨nchen, Germany

Accepted 6 October 1999

Abstract

The pharmacological properties of the expressed murine T-typeα1G channel were characterized using the whole cell patch clampconfiguration. Ba2+ or Ca2+ were used as charge carriers. Both IBa and ICa were blocked by Ni2+ and Cd2+ with IC50 values of0.47±0.04 and 1.13±0.06 mM (Ni2+) and 162±13 and 658±23 µM (Cd2+), respectively. Ni2+, but not Cd2+, modified the gating ofchannel activation. Ni2+ consistently accelerated channel deactivation while Cd2+ had a similar effect only on ICa. The α1G channelwas potently blocked by mibefradil in a dose- and voltage-dependent manner. IBa was moderately blocked by phenytoin (IC50

73.9±1.9 µM) and was resistant to the block by valproate. Also 3 mM ethosuximide blocked 20 and 35% of the IBa at a HP of2100 and260 mV, respectively, while 5 mM amiloride inhibited IBa by 38% and significantly slowed current activation. Theα1G

channel was not affected by 10µM tetrodotoxin. Both 1µM (+)isradipine and 10µM nifedipine inhibited 18 and 14% of IBa

amplitude at a HP of2100 mV, and 23% and 29% of IBa amplitude at a HP of260 mV, respectively. Theα1G current wasminimally activated by 1µM Bay K 8644. 2000 Elsevier Science Ltd. All rights reserved.

Keywords:T-type calcium channel; Mibefradil; Nickel; Cadmium; Antiepileptics; Dihydropyridines

1. Introduction

Epilepsies are common disorders affecting up to 1%of the caucasian population. More than 40 distinct formsof epilepsy have been identified. Low voltage activated(LVA) calcium channels have been implicated in thegeneration of a subform of epileptic seizures known asabsence seizures (Huguenard and Prince, 1994; Tsakiri-dou et al., 1995). Intracerebral electrode recordings fromhumans suggested the involvement of thalamic and neo-cortical neurons in the spike wave discharge of absenceseizures. The thalamic neurons involved have large LVAcalcium currents (ICa), also known as T-type ICa, whichplay an amplifying role in thalamic oscillation.Importantly, the principal mechanism by which mostdrugs against absence seizures are thought to act is by

* Corresponding author. Tel.:+49-89-4140-3283; fax:+49-89-4140-3261.

E-mail address:[email protected] (L. Lacinova´).1 On leave from Institute of Molecular Physiology and Genetics,

Slovak Academy of Sciences, Vlarska 5, 833 04 Bratislava, Slovakia.

0028-3908/00/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved.PII: S0028-3908 (99)00202-6

inhibition of the T-type current (Macdonald andKelley, 1995).

Recently, three genes (α1G, α1H andα1I) encoding theα1 subunit of LVA calcium channels have been ident-ified (Perez-Reyes et al., 1998; Cribbs et al., 1998; Leeet al., 1999; Klugbauer et al., 1999). Theα1G subunit ishighly expressed in the thalamus, cerebellum, hippocam-pus, olfactory bulb and neocortex (Klugbauer et al.,1999; Talley et al., 1999).

Pharmacological studies of the block of LVA ICa

yielded very variable results in native neuronal tissues.In different preparations the sensitivity to putative T-type calcium channel blockers often differed by 2–3orders of magnitude (reviewed in Huguenard, 1996).This variable affinity for antiepileptic drugs could becaused by a neuron specific expression of the three mem-bers of the cloned LVA channels or by the associationof different auxiliary subunits.

This study is an initial attempt to characterize thepharmacological profile of expressed LVAα1G channelsand to investigate the potential correlation between anti-absence seizure activity and inhibition of theα1G currentby antiepileptic drugs. We used the cloned murineα1G

1255L. Lacinovaet al. / Neuropharmacology 39 (2000) 1254–1266

subunit which encodes a predominantly neuronallyexpressed T-type calcium channel (Klugbauer et al.,1999). Upon functional expression in the HEK 293 cellline, we tested its modulation by a range of pharmaco-logical agents previously tested on T-type calcium chan-nels in various native tissues. It was found that theα1G

channel has a higher sensitivity to Cd2+ than to Ni2+.Furthermore, theα1G current is highly sensitive to mibef-radil, moderately sensitive to phenytoin, has a low sensi-tivity to ethosuximide, valproate, amiloride, dihydropyri-dine (DHP) blockers and activators, and is insensitive toTTX. These results suggest that theα1G subunit encodesa current that has only part of the well characterizedpharmacological properties of the native T-type channel.

2. Materials and methods

2.1. Transfection of HEK 293 cells

The full-length cDNA for the murineα1G subunit(Klugbauer et al., 1999) was cloned into the pcDNA 3vector (Invitrogen, San Diego, CA). HEK 293 cells weretransfected by lipofection with LipofectAMINE(Gibco BRL, Life Technologies, Germany).

2.2. Electrophysiological recordings

Ionic currents of transfected cells were recorded inwhole-cell configuration of the patch clamp method. Theextracellular solution contained (in mM): N-methyl-D-glucamine, 110; BaCl2 or CaCl2, 20; CsCl, 5; MgCl2, 1;HEPES, 10; glucose, 10; pH 7.4 (HCl). When Ba2+ wasused as a charge carrier, 0.2 mM EGTA was added tothe bath solution to eliminate Ca2+ contamination. Inexperiments with divalent cations (Ni2+, Cd2+) EGTAwas omitted from the Ba2+-based solution. The intra-cellular solution contained (in mM): CsCl, 102; MgATP,5; NaCl2, 5; TEACl2, 10; EGTA, 10; HEPES, 10; pH7.4 (CsOH). Currents were recorded using EPC-9 patchclamp amplifier and Pulse 8.05 software from HEKAElectronics (Lambrecht, Germany). Patch pipettes werepulled from borosilicate glass. The pipette input resist-ance was typically between 1.8 and 2.2 MV. The capaci-tance of individual cells ranged between 15 and 90 pFand series resistance ranged between 3.0 and 5.0 MV.Capacity transients were compensated using the built-inprocedure of the HEKA system. Series resistance wascompensated by up to 60%. Drug effects were investi-gated using trains of 40 ms long pulses applied fromholding potentials (HP) of2100 or 260 mV (asspecified) to210 mV with a frequency of 0.2 Hz. Cur-rent inhibition by mibefradil was also investigated at afrequency of 1 Hz. Current–voltage relations were meas-ured by a series of 15, 40-ms long depolarizing pulses

applied from HP of2100 mV to membrane potentialsbetween260 and+80 mV with a frequency of 0.33 Hz.For a steady state inactivation curve, the following pro-tocol was applied each 10 s: the cell was conditioned bya 5-s long pulse with amplitude between2120 and210mV, repolarized for 5 ms to a HP of2100 mV and thentested by 40-ms test pulses to210 mV. Tail currentswere analyzed during 20-ms long repolarizations to240mV after the 10-ms long test pulse from HP of2100 to210 mV (peak of current–voltage relationship). Currentamplitude was stable under described conditions withvirtually no run-down.

2.3. Data analysis

Data were acquired at 10 kHz and filtered at 2.9 kHz.Measured traces were not leak subtracted for the fittingof time courses of deactivation. Otherwise, leak currentwas subtracted using the P/4 procedure. The significanceof observed differences was evaluated by paired or non-paired Student t-tests, as appropriate. A probability of5% or less was considered to be significant. All experi-mental values are given as mean±SEM.

2.4. Solutions and drugs

All chemicals were of the highest purity available.(+)isradipine and Bay K 8644 were kind gifts from DrHof, formerly Sandoz AG and Dr Bechem, Bayer AG,respectively. Charged dihydropyridine (DHPch) wascustom-synthesized DHP analog, in which the activemoiety was linked to the charged [–+N(CH3)3] headgroup by an alkyl spacer chain containing 10 methylene(–CH2) groups (Bangalore et al., 1994). This length ofspacer chain was shown to optimize current blockingability of DHPch in native cardiac L-type calcium chan-nels (Bangalore et al., 1994). Mibefradil was a kind giftfrom Dr Clozel, Hoffmann-LaRoche (Basel,Switzerland). All other drugs were purchased fromSigma.

Stock solutions of DHPch (1 mM), TTX (1 mM) andmibefradil (10 mM) were prepared in water. Valproatewas dissolved directly in the bath solution. Stock sol-utions of (+)isradipine (10 mM), Bay K 8644 (10 mM),ethosuximide (100 mM) and phenytoin (50 mM) wereprepared in ethanol. Final concentration of ethanol inexperimental solutions was 0.1µl/ml for (+)isradipineand Bay K 8644, 1µl/ml for nifedipine, 2 µl/ml forphenytoin and 30µl/ml for ethosuximide.

Stock solution of amiloride (500 mM) was preparedin DMSO. The highest concentration of DMSO inexperimental solution was 10µl/ml.

Because of the relatively high concentration of vehiclein certain experiments, we tested the effects of ethanoland DMSO on expressedα1G channel. Both vehiclesinhibited the current throughα1G at high concentrations

1256 L. Lacinovaet al. / Neuropharmacology 39 (2000) 1254–1266

without affecting its kinetics. Threshold concentrationsfor current inhibition were 5 and 10µl/ml for ethanoland DMSO, respectively. Current inhibitions by etho-suximide and amiloride were therefore corrected forinhibition by the vehicle itself.

A control solution as well as drug-containing solutionswere applied in close proximity to the measured cells bylocal solution exchanger. Cells were placed near themouth of polyethylene tubing with an inner diameter of0.58 mm. The dead volume of the tip was approximately5 µl and the speed of the solution flow was approxi-mately 5µl/s. The new solution reached the investigatedcell in about 1 s.

3. Results

3.1. Block ofa1G channel by divalent cations

T-type calcium channels have previously been ident-ified according to their putative higher sensitivity to Ni2+

than to Cd2+. However, the IC50 for Ni2+ inhibition isreported to be in the range beween 30 and 780µM(reviewed in Huguenard, 1996) depending on the tissueinvestigated. Further complications may arise from thewide range of kinetic effects which Ni2+ has on variousHVA calcium channels (Zamponi et al., 1996). Wetherefore carefully compared the effects of Ni2+ andCd2+ on IBa and ICa of the expressedα1G channel.

Both IBa and ICa were inhibited by Ni2+ and Cd2+ inthe range from 10µM to 10 mM (Fig. 1A and Table 1).Cd2+ is a more effective channel blocker than Ni2+ andboth divalent cations inhibited IBa more effectively thanICa. Cd2+, but not Ni2+, shifted the reversal potential forIBa significantly by213.6 mV (compare Figs 1B–E). Incontrast, Ni2+, but not Cd2+, significantly affected thevoltage dependence of both IBa and ICa activation (Fig.2 and Table 2).

To analyze the effects of divalent cations on the timecourse of current through theα1G channel, we fitted indi-vidual current traces measured during current–voltagerelation protocol (see insets to Fig. 2) using the m4hform of the Hodgkin–Huxley equation. Cd2+ had no sig-nificant effect on the time constants of current activationor inactivation, and this was independent of the chargecarrier (data not shown). Ni2+ did not significantly affectthe activation time constants of IBa or ICa nor the inacti-vation time constant of ICa (not shown). However, 1 mMNi2+ significantly decreased the inactivation time con-stant of IBa at depolarizing pulses to220 mV (p,0.05),0 mV (p,0.01) and+10 mV (p,0.01).

Although the divalent cations had little effect on theactivation and inactivation rate, they significantly affec-ted the deactivation rate of the channel. Both Ni2+ andCd2+ accelerated the deactivation of ICa in a concen-

tration-dependent manner when the channel was acti-vated by a 10 ms depolarizing pulse to210 mV fol-lowed by a 20 ms pulse to240mV (Figs 3A–C). Ni2+,but not Cd2+, also accelerated deactivation of IBa (Figs3D–F). Since the same concentrations of Ni2+ and Cd2+

caused a different inhibition of the current amplitude, wecompared the dependence of current deactivation kin-etics to the extent of amplitude inhibition. While thesemeasurements virtually overlapped for both divalent cat-ions when Ca2+ was used as a charge carrier, they dif-fered strongly when Ba2+ was used as a charge carrier(Figs 3B and E).

In summary, while the inhibition of theα1G channelby Cd2+ and Ni2+ exhibited some common features, anumber of differences were observed that suggest a morespecific interaction of the channel with Ni2+ ions.

3.2. Block of the current througha1G channel bymibefradil

Mibefradil was introduced as a calcium channelblocker with selectivity for the T-type current (forreview see Clozel et al., 1997). This drug interacts withthe native T-type calcium channels and with allexpressed HVA channels in a state-dependent manner(Mehrke et al., 1994; Bezprozvanny and Tsien, 1995;Randall and Tsien, 1997). We have demonstrated thatIBa through the expressedα1G channel is inhibited bymibefradil with IC50 values of 0.39 and 0.12µM at hold-ing potentials (HPs) of2100 and260 mV, respectively(Klugbauer et al., 1999). To test if a conducting ion hasan influence on inhibition by mibefradil, we replacedBa2+ by Ca2+ as a charge carrier. The extent of amplitudeinhibition was not significantly altered. Here, 300 nMmibefradil blocked 34±4% of IBa (Klugbauer et al.,1999) and 38±2% of ICa (Fig. 4A). The time course ofblock development had a fast and a slow component.The fast component corresponds to the closed channelblock and was evaluated as an inhibition of the currentamplitude measured during the second pulse in the pres-ence of the drug. The slow component which developsduring repetitive pulsing corresponds to the use-depen-dent block. This component may be fitted by a singleexponential with time constant of 103 s. When the HPwas shifted to260 mV, at which 30% of all channelsare inactivated (Klugbauer et al., 1999), the initial blockincreased significantly from 14±2% to 36±5% (Fig. 4B).The use-dependent block was also accelerated with atime constant of 32 s.

Recovery from mibefradil inhibition during washoutof the drug had two exponential components in eachcase. The time constants were 13 and 87 s at a HP of2100 mV, and 15 and 127 s at a HP of260 mV. Thisslight enhancement is probably less important than theobserved decrease in the on-rate for the voltage-depen-dent character of inhibition.


Fig. 1. Block of α1G channel by Ni2+ and Cd2+. (A) Dose–response curves for the inhibition of IBa (closed symbols) and ICa (open symbols) byNi2+ (P,s) and Cd2+ (j,h), respectively. Solid lines represent fits of experimental points by the Hill equation. Results of fitting procedures aresummarized in Table 1. (B) Current–voltage relations for ICa measured under control conditions (s) and in the presence of 1 mM Ni2+ (P). (C)Current–voltage relations for IBa measured under control conditions (s) and in the presence of 1 mM Ni2+ (P). (D) Current–voltage relations forICa measured under control conditions (h) and in the presence of 1 mM Cd2+ (j). (E) Current–voltage relations for ICa measured under controlconditions (h) and in the presence of 0.3 mM Cd2+ (j).


Table 1IC50 values for inhibition of IBa and ICa by Ni2+ and Cd2+. The experimental data of Fig. 1A were fitted to the Hill equation, then number of cellstested is given in brackets

ICa IBa

IC50 [µM] n IC50 [µM] n

Ni2+ 1130±60 (6–11) 1.17±0.07 470±40 (6–11) 1.07±0.04Cd2+ 658±23 (7–9) 0.93±0.03 162±13 (6–9) 0.95±0.07

Fig. 2. Shift of the voltage dependence of current activation by Ni2+ but not by Cd2+. G was calculated from the experimental data shown inFig. 1 asG=I/(Vmembr2Vrev), whereI represents the current amplitude,Vmembr is the membrane potential of the corresponding depolarizing pulseandVrev is the reversal potential. IndividualG–V relations were normalized to the maximal conductance and averaged. Solid lines represent fits ofexperimental data by the Boltzmann equation. Results of the fitting procedures are summarized in Table 2. Activation curves for ICa (A) and IBa

(B) measured in the absence (s) and presence of 1 mM Ni2+ (P). Activation curves for ICa (C) and IBa (D) measured in the absence (h) andpresence of 1 mM (C) and 0.3 mM (D) Cd2+ (j). In each panel, the current traces recorded during the whole current–voltage relation protocolare shown as insets. Open symbols represent control conditions, filled symbols describe recordings in the presence of the corresponding cation.Scale bars are common for recordings under both experimental conditions.

Table 2Effects of Ni2+ and Cd2+ on voltage dependence of activation of IBa and ICa. Data are results of fits of the conductance–voltage curves shown inFig. 2 by the Boltzmann equation. Then number of cells tested is given in brackets. Asterisks mark statistically significant differences betweenvalues measured in the presence of the divalent cation and the corresponding control value. *p,0.05, *** p,0.001 in paired Student’s t-test

V0.5 (mV) k (mV)Control Cation Control Cation

ICa/1 mM Ni2+ 222.2±1.6 (7) 217.8±1.6*** 4.1±0.4 5.0±0.3***IBa/1 mM Ni2+ 224.2±0.9 (7) 215.6±0.8*** 4.6±0.3 6.4±0.4***ICa/1 mM Cd2+ 220.6±1.0 (7) 219.6±1.6 4.3±0.4 5.1±0.5*IBa/0.3 mM Cd2+ 224.0±1.4 (6) 225.6±1.1 4.6±0.4 4.6±0.5


Fig. 3. Acceleration of ICa and IBa deactivation by Ni2+ and Cd2+. (A) Time constants of ICa deactivation measured at different concentrations ofNi2+ (s, n=6) and Cd2+ (h, n=5). (B) Relations between the ability of Ni2+ (s) and Cd2+ (h) to inhibit ICa amplitude and to accelerate the timeconstant of ICa deactivation. (C) Examples of tail currents recorded with Ca2+ as a charge carrier under control conditions and in the presence ofvarious concentrations of Ni2+ (upper) or Cd2+ (bottom). (D) Time constants of IBa deactivation measured at different concentrations of Ni2+ (P,n=3–10) and Cd2+ (j, n=5). (E) Relations between the ability of Ni2+ (P) and Cd2+ (j) to inhibit ICa amplitude and to accelerate the time constantof ICa deactivation. (F) Examples of tail currents recorded with Ba2+ as a charge carrier under control conditions and in the presence of variousconcentrations of Ni2+ (upper) or Cd2+ (bottom). For the pulse protocol see paragraph 2.2 in Materials and methods.

When the pulse frequency was increased to 1 Hz, thecurrent amplitude decreased by 10% in the absence ofthe drug due to accumulation of the channels in theirinactivated state (Fig. 4C). In the presence of mibefradilonly the use-dependent component of the block with atime constant of 9 s was observable. When inhibition bymibefradil was compared with the block whichdeveloped during the same number of consecutive pulsesat 0.2 Hz (Fig. 4D), it became apparent that channel inhi-bition depended on the number of channel depolariza-tions and not on their frequency.

In all three cases, the time course of ICa during thedepolarizing pulse was not affected by the presence ofthe drug as is shown in representative examples of cur-rent traces in Figs 4A–C.

3.3. Moderate sensitivity of the expresseda1G channelto phenytoin

Phenytoin is an anticonvulsant which is reported toblock T-type calcium currents in a neuroblastoma cellline (Twombly et al., 1988), in rat hippocampal neurons(Yaari et al., 1987), rat thalamic neurons (Coulter et al.,1989a) and in rat sensory neurons (Todorovic and Lin-gle, 1998) at concentrations between 1 and 100µM.

Phenytoin blocked IBa of the expressedα1G in a concen-tration-dependent manner with an approximate IC50 of73.9±1.9 µM and a Hill coefficient of 1.06±0.03 (n=7)(Fig. 5A). The current kinetics were not influenced bythe presence of the drug (see inset to Fig. 5A) and theblock was independent of the membrane potential of thetest pulse (Figs 5B and C).

3.4. Low sensitivity ofa1G channel to ethosuximide,valproate, amiloride and TTX

In addition to phenytoin, we tested the effect of twoother antiepileptic drugs, ethosuximide and valproate,reported to act by inhibition of neuronal T-type calciumchannels. IBa was inhibited 15.3±2.9% by 3 mM of etho-suximide at a HP of2100 mV. A shift of the HP by+40 mV significantly increased this inhibition to34.2±5.6% (Fig. 6A). The current kinetics were notaffected by ethosuximide (Fig. 6B). The significant volt-age dependence of the block suggested that the interac-tion of ethosuximide with the channel is specific. Valp-roate inhibited the IBa slightly at concentrations above10 µM, reaching inhibition of 10.5±2.5% at 1 mM (Fig.6A, data for lower concentrations not shown).

Amiloride, a blocker with a reported moderate affinity


Fig. 4. Mibefradil blocks theα1G channel in a voltage-dependent manner. (A) Time course of the inhibition of ICa amplitude by 300 nM mibefradilmeasured from a HP of2100 at a pulse frequency of 0.2 Hz. Results from nine cells were averaged. Solid lines are the fits of experimental pointsto the sum of two exponentials. Insets show current traces before (s) and at the end (P) of mibefradil application, either raw or scaled to thesame amplitude. Scale bars mark 10 ms (horizontal) and 500 pA (vertical). (B) Time course of the inhibition of ICa amplitude by 300 nM mibefradilmeasured from a HP of260 at a pulse frequency of 0.2 Hz. A total number of eight cells were averaged. Key to the symbols are the same as inpanel (A). Scale bars represent 10 ms and 500 pA. (C) Time course of inhibition of ICa amplitude by 300 nM mibefradil measured from a HP of2100 at a pulse frequency of 1 Hz. Data from seven cells were averaged. Same symbols as in panel (A). Scale bars represent 10 ms and 200 pA.(D) Comparison of the time courses of mibefradil effect from the experiments in panels (A) and (C). Current amplitude is plotted against the pulsenumber and not against time.

Fig. 5. Block of theα1G channel by phenytoin. (A) Dose–response curve for phenytoin measured from a HP of2100 mV. Current traces in theabsence (s) and presence (P) of 100 µM phenytoin are shown in the inset. The solid line represents the fit of the experimental points by the Hillequation with an IC50 of 73.9±1.9 µM and a Hill coefficient ofn=1.06±0.03. Between five and seven cells were tested at each drug concentration.(B) Current amplitudes measured from a HP of2100 mV by depolarizing pulses with amplitude as marked in the absence (s) and presence of100 µM phenytoin (P). (C) Relative amplitude of the current in the presence of 100µM phenytoin measured at membrane potentials as marked(calculated from data in panel (B)).


Fig. 6. Effect of ethosuximide, amiloride and TTX on the current through theα1G channel. (A) Block diagram showing relative current amplitudemeasured in the presence of each drug at the indicated concentrations as a % of control current amplitude. Number of cells tested was seven(ethosuximide, HP=2100 mV), five (ethosuximide, HP=260 mV), six (valproate, 1 mM), seven (amiloride, 1 mM), eight (amiloride, 5 mM) andfive (TTX). ** p,0.01 between the effects of ethosuximide at HPs of2100 and260 mV (unpaired t-test). (B) Current records in the absenceand presence of 3 mM ethosuximide, as marked, measured from a HP of2100 mV (left) or260 mV (right). (C) Current records measured underthe control conditions (s), in the presence of 1 mM (left) or 5 mM (right) amiloride (P) and after washout (h) of the drug. Inset in the rightpart of the panel shows the activation of the current on an expanded time scale and at a normalized amplitude to facilitate the comparison ofcurrent kinetics.

to T-type calcium channels (Tang et al., 1988; Todorovicand Lingle, 1998; Arnoult et al., 1998), inhibited theα1G

channel at millimolar concentrations (23.1±1.6 and38.0±2.4% inhibition at concentrations of 1 and 5 mM,respectively). In addition to a decrease in current ampli-

tude, the kinetics of current activation were significantlyslowed (inset to Fig. 6C). The 10–90% growth time forthe current during depolarization from a HP of2100 to210 mV was 1.79±0.17 ms in the absence and2.31±0.11 ms in the presence of 5 mM amiloride (n=7;


p,0.01 in paired t-test). TTX at a concentration of 10µM failed to affect the time course and/or the amplitudeof IBa through the expressedα1G channel.

3.5. Modulation ofa1G channel by dihydropyridines

DHPs are L-type calcium channel blockers and acti-vators. Several authors have suggested that they maymodulate T-type calcium channels at higher concen-trations (for review see Vassort and Alvarez, 1994). TheL-type calcium channel blocker isradipine inhibits L-type calcium channels in the nM concentration range ina highly voltage-dependent manner. A thousand foldhigher concentration of isradipine (1µM) inhibited IBa

through theα1G channel by only 18.2±1.7%. This inhi-bition was slightly enhanced to 23.3±3.0% when the HPwas shifted by+40 mV (Fig. 7A). Isradipine did notaffect the time course of the current (Fig. 7B). Nifedipine(10µM) inhibited 14.0±1.7 and 28.7±2.8% of the currentamplitude at HP values of2100 and260 mV, respect-ively. A more depolarized HP significantly increased theblocking efficiency of nifedipine (Fig. 7A). In addition,the current was affected slightly by 1µM Bay K 8644,an L-type calcium channel agonist. The rising part of thecurrent–voltage relation (Fig. 7C) was shifted by24mV, resulting in an increased current amplitude at depol-arizing pulses to230 and220 mV. The current inacti-vation was accelerated in the presence of Bay K 8644(Fig. 7D), but the time course of current deactivationwas not altered.

Recently, we have shown that DHPch, a custom-syn-thesized DHP analog with a charged head group (seeMaterials and methods), interacts differently with the L-type α1C calcium channel than with neutral DHPs(Lacinovaet al., 1999). Therefore, we tested whether theT-type calcium channel has retained this putativeDHPch-site. DHPch inhibited the expressed L-typeα1C

calcium channel with an IC50 of 170 nM at HP of280mV. The inhibition was only weakly voltage dependent(Lacinovaet al., 1999). DHPch was found to be a weakblocker of the expressedα1G channel; 1µM of the druginhibited 22.0±3.9% (n=4) of current amplitude (Fig.7E). When the HP was shifted by+40 mV, the inhibitionincreased to 37.5±5.9% (n=6), but this enhancement wasnot statistically significant (unpaired t-test). The lack ofvoltage dependence of the block was further supportedby the small effect of DHPch on the steady-state inacti-vation curve (Fig. 7F).

4. Discussion

In this paper we describe the characterization of theinteraction of the expressedα1G channel with severalreported organic and inorganic T-type channel blockers.

The α1G channel is expressed abundantly in the brainand to a lower extent in the heart (Perez-Reyes et al.,1998; Klugbauer et al., 1999). The predominant T-typecalcium channel in the heart appears to be theα1H chan-nel, another member of the LVA-channel family (Cribbset al., 1998; Williams et al., 1999). In the brain theα1G

channel is expressed predominantly in the thalamus,hypothalamus, midbrain, cerebellum and medulla, whereits expression dominates over the expression of theα1H

and α1I channels (Talley et al., 1999).

4.1. Inorganic channel blockers

The expressedα1G channel has a similar sensitivityfor Cd2+ as the expressedα1H channel (IC50 162 µMversus 104µM; this work and Williams et al., 1999).However, both channels differ strongly in their sensi-tivity to Ni2+. While the IC50 for IBa of the α1G channelis 470µM, the IC50 is only 6.6µM for the α1H channel(Williams et al., 1999). This difference, together withthe differential expression of both channels, may explainthe inconsistencies in the reported Ni2+-sensitivity ofnative T-type calcium currents.

The interaction of Ni2+ ions with theα1G channel wasfound to alter the gating parameters of the channel. Ni2+

shifted the voltage of activation by+10 mV andincreased the activation slope parameter. Replacement ofBa2+ by Ca2+ as the charge carrier decreased the affinityfor Ni2+. Similar changes have been observed when theneuronal HVA calcium channelsα1A, α1B andα1E wereused (Zamponi et al., 1996). The time course of acti-vation of theα1G channel was not altered by Ni2+, incontrast to its effect on the HVA L-type calcium chan-nels (McFarlane and Gilly, 1998). On the other hand,acceleration of channel deactivation by Ni2+ was notseen in HVA channels (Zamponi et al., 1996; McFarlaneand Gilly, 1998). The effect of Ni2+ on channel gatingwas not mimicked by the other divalent inorganic cation,Cd2+. Apparently, Ni2+ interacts in an exclusive and spe-cific way with theα1G channel. These features could beused as a tool to identify theα1G current in native tissue.

4.2. Organic channel blockers

Mibefradil was developed as a specific blocker of T-type calcium channels (reviewed in Clozel et al., 1997).We confirmed this specificity, observed in native tissues,for the expressed channel. Expressed HVA calciumchannels have an IC50 for inhibition by mibefradil ofbetween 3.1 and 21µM at hyperpolarized membranepotentials (Bezprozvanny and Tsien, 1995; Lacinova´ etal., 1995). Expressedα1G channel was 10–50-fold moresensitive than HVA channels at a hyperpolarized mem-brane potential and slightly more sensitive than theexpressedα1H channel (Cribbs et al., 1998; Williams etal., 1999; Klugbauer et al., 1999). The blocking potency


Fig. 7. Effect of dihydropyridines on theα1G channel. (A) Relative amplitude of IBa as a % of control current amplitude in the presence of 1µM (+)isradipine measured from a HP of2100 mV (n=5) or 260 mV (n=4) and 10µM nifedipine measured from a HP of2100 mV (n=8) or260 mV (n=7). (B) Examples of currents recorded under control conditions (s) and in the presence of 1µM (+)isradipine measured from a HPof 2100 mV (upper) or260 mV (lower). (C) Averaged current–voltage relations measured in the absence (s) and presence (P) of 1 µM BayK 8644 (n=5 cells). ** p,0.01; *** p,0.001 in paired t-test. (D) Examples of currents recorded under control conditions (s) and in the presenceof 1 µM Bay K 8644 (P) during depolarizing pulse to220 mV. In the lower part of the diagram, both traces were scaled to the same amplitudeto facilitate comparison of current kinetics. (E) Relative amplitude of IBa as a % of control current amplitude in the presence of 1µM DHPchmeasured from a HP of2100 mV (n=4) or 260 mV (n=6). (F) Steady-state inactivation measured in the absence (s, n=5) and presence (P, n=5)of 1 µM DHPch. The inset shows current recordings measured in the absence (s) and presence (P) of 1 µM DHPch at a HP of2100 mV (upper)or 260 mV (bottom).


of the drug is not influenced by the charge carrier used(Ba2+ or Ca2+). The sensitivity ofα1G to mibefradil andthe observed voltage dependence of the block are lesspronounced than that reported by McDonough and Bean(1998) in cerebellar Purkinje neurons, whereα1G is parti-cularly abundant (Talley et al., 1999). Our finding thatthe voltage-dependent character of the inhibition iscaused by an increased on-rate rather than by a decreasedoff-rate also contrasts with this report.

Mibefradil is a much more potent blocker ofα1G chan-nel than any other organic blocker tested in this report.Amiloride, a commonly used blocker of Na+/Ca2+ andNa+/H+ exchangers, has been reported to block Ca2+

channels with an apparent selectivity for the T-typechannels. Its reported IC50 values vary from 75µM inrat sensory neurons (Todorovic and Lingle, 1998) to 0.5mM in rat trigeminal root ganglion cells (Kim andChung, 1999). The expressedα1G channel is even lesssensitive, with an apparent IC50 above 5 mM. Amilorideinteracts with the channel at a specific site as inferredfrom its effect on channel activation. It could be thatother members of the LVA channels family and/or anunidentified regulatory factor are needed to produce highamiloride sensitivity in sensory neurons.

The basic therapeutic properties of established antiepi-leptic drugs is thought to be due to a decrease in mem-brane excitability of neurons that results from the inter-action of the drug with neurotransmitter receptors or ionchannels. It is proposed that this interaction involves theblock of T-type calcium or sodium currents. The thera-peutic concentration of phenytoin is 4–8µM (Macdonaldand McLean, 1986). This concentration was shown toreduce T-currrent amplitude by 10–30% in the thalamus(Coulter et al., 1989a) and in sensory neurons(Todorovic and Lingle, 1998). In a neuroblastoma cellline (Twombly et al., 1988) and in rat hippocampal neu-rons (Yaari et al., 1987) 100µM of phenytoin was neces-sary to inhibit 50% of the T-current amplitude.Expressedα1G channel in this study is blocked by about15% by the clinically relevant concentration of pheny-toin. Because the T-type current contributes to the rapidonset of the action potential, the inhibition of theα1G

channel could contribute to the therapeutic effects ofphenytoin.

The therapeutic concentration of ethosuximide is inthe range 300–700µM (Macdonald and McLean, 1986).Within this concentration range, ethosuximide wasshown to inhibit up to 40% of T-type calcium currentamplitude in thalamic neurons (Coulter et al., 1989b,1990) but had no effect in human temporal neocortex(Sayer et al., 1993) and in rat thalamic neurons (Lerescheet al., 1998). In contrast, 24 mM ethosuximide wasnecessary to reach half-maximal inhibition in rat sensoryneurons (Todorovic and Lingle, 1998). The sensitivityof the expressedα1G corresponds to that reported byTodorovic and Lingle (1998). Nevertheless, the observed

voltage dependence suggests that this low affinity inter-action represents a genuine drug–receptor interaction andnot just a non-specific effect.

Valproate was shown to inhibit up to 17% of LVAcurrent amplitude in the sensory neurons with an IC50

of 330µM (Todorovic and Lingle, 1998). In human tem-poral neocortex valproate was without effect (Sayer etal., 1993). The therapeutically relevant concentrationsrange from 6 to 200µM (Macdonald and McLean,1986). This range corresponds to the threshold for aninhibitory effect of valproate on the expressedα1G chan-nel. Although the observed inhibition level is low, thisinhibition might be clinically important to suppressrepetitive firing.

Dihydropyridines are known as high affinity antagon-ists and agonists of L-type calcium channels. In addition,interactions of DHPs with the T-type calcium currentwere documented by several authors. Antagonistic DHPswere reported to inhibit the T-type current in concen-trations between 100 nM (Kuga et al., 1990; Arnoult etal., 1998) and 10µM (Richard et al., 1991). Theexpressedα1G current belongs to the group with a lowaffinity to antagonistic DHPs. Bay K 8644, a DHP agon-ist of the L-type calcium channels, acts as an antagonistof neuronal T-type calcium channels (Richard et al.,1991; Akaike et al., 1989) or potentiates the T-currentby 100% at submicromolar concentrations (Stengel etal., 1998). The expressedα1G current is not blocked byBay K 8644 in this study. Bay K 8644 had a weak agon-istic effect on theα1G channel at negative membranepotentials suggesting that we cannot reproduce thereported in vivo effects with theα1G channel.

DHPch, a dihydropyridine-type blocker with acharged head, inhibits theα1C channel with an IC50 of170 nM (Lacinova´ et al., 1999). A 50% inhibition ofexpressedα1G channel required more than 1µM DHPch,suggesting at least a 10-fold higher IC50. Theα1G chan-nel therefore probably does not contain the putativeinteraction site described for theα1C channel (Lacinova´et al., 1999).

A TTX-sensitive low-voltage activated Ca2+ currenthas been identified in cardiac and neuronal preparations(Aggarwal et al., 1997; Balke et al., 1999). The kineticsof this channel resembled that of a T-type calcium chan-nel. The current was insensitive to low concentrations ofNi2+ and was inhibited by 10µM of TTX. The α1G chan-nel shares some features with this channel. However, dueto its insensitivity to TTX, theα1G channel cannot con-tribute to this type of calcium conductance in nativetissues.

In summary, we have characterized the interactions ofthe expressedα1G channel with a series of inorganic andorganic channel blockers. In contrast to many reports onthe T-type calcium current in native tissues, theexpressed channel has a relatively low sensitivity to mostof the tested organic blockers suggesting that other mem-


bers of the LVA channel family and/or unidentified regu-latory factors contribute to many of the reported effects.On the other hand, theα1G channel interacts in a highlyspecific manner with the inorganic divalent cation Ni2+

and has many of the properties of the neuronal T-typecalcium channel, inhibition of which contributes to thetherapeutic effect of drugs for absence epilepsy.

Acknowledgements

This work was supported by the Deutsche Forschung-sgemeinschaft and Fond der Chemie.

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regulation of the calcium channel α1g subunit by divalent cations and organic blockers

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