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1-subunit and 
-subunit interaction affect sensitivity for the phenylalkylamine (-)gallopamil
MS 9496 Received 8 April 1999; accepted after revision 2 June 1999.
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ABSTRACT |
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1A-PAA). Use-dependent barium current (IBa) inhibition of 1A-PAA by (-)gallopamil and Ca2+ channel recovery from inactivation and block were studied with two-microlectrode voltage clamp after expression of 1A-PAA and auxiliary 2-
- and 1a- or 2a-subunits in Xenopus oocytes.
1A numbering) in the intracellular loop connecting domains I and II of 1A-PAA slowed the inactivation kinetics and reduced use-dependent inhibition (100 ms test pulses at 0·2 Hz from -80 to 20 mV) of the resulting mutant 1A-PAA/R-E/1a channels by 100 µM (-)gallopamil (53 ± 2 %, 1A-PAA/1a vs. 31 ± 2 %, 1A-PAA/R-E/1a, n 
4). This amino acid substitution simultaneously accelerated the recovery of channels from inactivation and from block by (-)gallopamil.
1A-PAA with the 2a-subunit reduced fast IBa inactivation and induced a substantial reduction in use-dependent IBa inhibition by (-)gallopamil (25 ± 4 %, 1A-PAA/2a; 13 ± 1 %, 1A-PAA/R-E/2a). The time constant of recovery from block at rest was not significantly affected.
2a-1-subunit interaction affect the drug-channel interaction.
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INTRODUCTION |
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Calcium (Ca2+) channel inhibition by drugs such as phenylalkylamines (PAAs), benzothiazepines (BTZs) and mibefradil increases during repetitive depolarisation of the membrane (Lee & Tsien 1983; McDonald et al. 1984; Bezprozvanny & Tsien 1995; Aczél et al. 1998). Such a 'use-dependent' channel inhibition reflects distinct drug interactions with the resting, open and inactivated channel states. It is believed that state-dependent Ca2+ channel block plays an important role in the therapeutic action of PAAs and BTZs as antiarrhythmics (Hondeghem & Katzung, 1984).
Functional studies on mutant Ca2+ channels enabled the first insights into the molecular architecture of the Ca2+ channel drug-binding domains (see Hockerman et al. 1997b and Striessnig et al. 1998 for review). The available data suggest that three different classes of Ca2+ channel antagonists (PAAs, BTZs and 1,4-dihydropyridines) bind in close proximity within the pore region of L-type Ca2+ channel 1-subunits (Striessnig et al. 1998). Three amino acids in segment IVS6 (Tyr1463, Ala1467, Ile1470) and four residues in transmembrane segment IIIS6 (Tyr1152, Ile1153, Phe1164 and Val1165) have been identified as crucial L-type determinants of the PAA sensitivity (Hockerman et al. 1995, 1997a). Insertion of three 'L-type-specific' residues (Tyr1463, Ala1467 and Ile1470) into segment IVS6 of the only weakly PAA sensitive class A (1A) Ca2+ channel transferred PAA sensitivity to the corresponding 1A mutant (here called 1A-PAA; Hering et al. 1996).
An unequivocal identification of the PAA binding determinants by mutational analysis of 1 Ca2+ channel subunits is, however, complicated by an apparent interdependence between Ca2+ channel block and inactivation gating (see Hering et al. 1998 for review). In particular, transfer of the IVS6 L-type determinants of PAA sensitivity (Tyr1463, Ala1467 and Ile1470) to class A Ca2+ channels accelerated inactivation (Hering et al. 1996; Degtiar et al. 1997). Moreover, introduction of an additional L-type amino acid (Met1464 into IVS6 of 1A-PAA) facilitated channel inactivation and enhanced use-dependent channel block by (-)gallopamil (Hering et al. 1996). Accordingly, substitution of a PAA determinant in 1A-PAA (Ile1470 by the corresponding class A channel Met, 1C-a numbering) which substantially reduced channel inactivation induced an about 30-fold decrease of the apparent association rate for (-)devapamil (Degtiar et al. 1997) and alanine substitutions of three L-type amino acids localised close to the inner channel mouth on segment IIIS6 and IVS6 reduced Ca2+ channel inactivation and simultaneously BTZ and PAA sensitivity (Hering et al. 1997; Berjukow et al. 1999).
In our previous studies on the role of Ca2+ channel inactivation in channel block by PAAs and BTZs we have focused on residues that are located on segments IIIS6 and IVS6 (Degtiar et al. 1997; Hering et al. 1997; Berjukow et al. 1999). Here we analyse in a PAA-sensitive class A Ca2+ channel mutant (1A-PAA) expressed in Xenopus oocytes if inactivation determinants localised outside the channel pore of an 1-subunit influence Ca2+ channel block by (-)gallopamil.
We demonstrate that a single amino acid substitution (Arg387Glu, 1A numbering) in the intracellular loop between domains I and II slows channel inactivation and reduces sensitivity for (-)gallopamil. Furthermore, a reduced inactivation caused by coexpression of 1A-PAA with 2a- instead of the 1a-subunit reduced Ca2+ channel block by (-)gallopamil even more dramatically. Our study clearly demonstrates that inactivation determinants that are localised outside the putative drug binding regions in the channel pore affect the molecular mechanism of use-dependent Ca2+ channel block by (-)gallopamil.
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METHODS |
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Generation of 1A-constructs
The construction of the PAA-sensitive triple rabbit brain class A Ca2+ channel mutant AL25 (named herein 1A-PAA) was previously described (Hering et al. 1996). The derived mutant 1A-PAA/R-E was constructed by introducing a single point mutation (R387E, 1A numbering) into 1A-PAA cDNA by the 'gene SOEing' technique (Horton et al. 1989). The point mutation was verified by sequence analysis. All constructs were inserted into the polyadenylating transcription plasmids pSPCBI 2 (a kind gift of Dr O. Pongs, University of Hamburg).
Electrophysiology
Female Xenopus laevis (NASCO, Fort Atkinson, WI, USA) were anaesthetised by exposing them for 15 min to a 0·2 % MS-222 (methane sulfonate salt of 3-aminobenzoic acid ethyl ester; Sandoz) solution before surgically removing parts of the ovaries. The frogs were then allowed to recover and returned to their tank. Each frog was reused up to two times and subsequently killed by decapitation under anaesthesia. The interval between the operations was longer than 4 months. Follicle membranes from isolated oocytes were enzymatically digested with 2 mg ml-1 collagenase (Type 1A, Sigma). Calcium channel currents (IBa) were studied 2 to 7 days after microinjection of approximately equimolar cRNA mixtures of 1 (0·3 ng per 50 nl)-1a(2a) (0·1 ng per 50 nl)-2 (0·2 ng per 50 nl) with two-microelectrode voltage clamp of Xenopus oocytes with 40 mM Ba2+ as charge carrier in a bath solution containing (mM): 40 Ba(OH)2, 40 N-methyl-D-glucamine, 10 Hepes, 10 glucose, adjusted to pH 7·4 with methanesulfonic acid as previously described (Grabner et al. 1996). Endogenous chloride currents of the oocytes were suppressed by injecting 20-40 nl of a 0·1 M BAPTA solution 30-240 min before the voltage clamp measurements. Voltage-recording and current-injecting microelectrodes were filled with 2·8 M CsCl, 0·2 M CsOH, 10 mM EGTA, 10 mM Hepes (pH 7·4) and had resistances of 0·3-2 M
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Drug sensitivity was estimated as use-dependent Ca2+ channel block during 20 test pulses (100 ms) applied at 0·2 Hz from -80 mV to 20 mV corresponding to the peak current voltage of the current-voltage relationships of all studied Ca2+ channel mutants. Use-dependent block was measured after a 3 min equilibration of the oocytes in drug-containing solution. To estimate the accumulation of Ca2+ channels in inactivation under control conditions similar pulse trains were applied in the absence of drug. Resting channel block was measured in an individual set of experiments as peak IBa inhibition during 100 ms test pulses from -80 to 20 mV after a 5 min equilibration in drug-containing solution.
Recovery from inactivation was studied at a holding potential of -80 mV after depolarising Ca2+channels during a 3 s prepulse to 20 mV by applying 30 ms test pulses to 20 mV at various time intervals after the conditioning prepulse. Peak IBa values were normalised to the peak current measured during the prepulse. The time course of IBa recovery from inactivation was fitted to a biexponential function:
fast) + Bexp(-t/slow) + C.
Initial rates of IBa decay (see Fig. 1B) were estimated by calculating the maximum derivative of mono- (1A-PAA/2a, 1A-PAA/R-E/2a) or biexponential (1A-PAA/1a, 1A-PAA/R-E/1a) fits to current inactivation during a 3 s depolarisation from -80 to 20 mV.
Voltage dependence of IBa inactivation ('inactivation curve') was measured as normalised peak current during a 30 ms test pulse that was applied 3 ms after a 3 s conditioning prepulse to a given voltage. Conditioning and test pulses were applied every 60 s from a holding potential of -100 mV. Inactivation curves were fitted to the equation:
Data are given as means ± S.E.M. Statistical significance was calculated according to Student's unpaired t test (P < 0·05).
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RESULTS |
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Mutation Arg387Glu in the domain I-II loop of the PAA-sensitive 1A-PAA-subunit and 2a-subunit interaction affect channel inactivation and sensitivity for (-)gallopamil
We have previously reported that amino acid substitutions in transmembrane segments IIIS6 and IVS6 of class A and class C 1-subunits affect Ca2+ channel inactivation kinetics and simultaneously sensitivity for PAA and BTZ (Hering et al. 1998). In order to further characterise the role of channel inactivation in Ca2+ channel block by PAAs we substituted arginine by glutamine in position 387 of the intracellular loop between domains I and II of the PAA-sensitive class A channel mutant 1A-PAA (see Herlitze et al. 1997).
As expected from previous studies on wild-type class A channels (Herlitze et al. 1997), the I-II loop mutation Arg387Glu reduced the rate of current decay of the resulting quadruple mutant 1A-PAA/R-E and shifted the mid-point of the inactivation curve to more positive potentials (Fig. 1A, B and F). IBa of 1A-PAA/R-E/1a displayed less use-dependent IBa inhibition by (-)gallopamil (10 and 100 µM) compared with 1A-PAA/1a (Fig. 1C and D).
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1-2a -subunit interaction reduce channel block by (-)gallopamil
A, normalised IBa of mutants | ||
Next we determined if a modulation of the current decay by different -subunits would affect Ca2+ channel block by (-)gallopamil. It is well established that coexpression of the 2a-isoform induces a slow rate of voltage-dependent inactivation of Ca2+ channels (Stea et al. 1994). Accordingly, coexpression of the mutants 1A-PAA and 1A-PAA/R-E with the 2a-subunit dramatically decreased IBa inactivation (Fig. 1A and B). Again, slower inactivation kinetics induced significantly less use-dependent block of 1A-PAA/2a and 1A-PAA/R-E/2a compared with 1A-PAA/1a and 1A-PAA/R-E/1a channels (Fig. 1C and D). Interestingly, peak IBa inhibition by (-)gallopamil occurred at a faster rate if Ca2+ channels were coexpressed with the 1a-subunit (Fig. 1E). There was little additional effect of the point mutation on inactivation rate once the 2a-subunit was coexpressed (Fig. 1A); however, there was still a substantial effect on drug sensitivity (Fig. 1D).
We did not observe significant resting channel block (<5 %) by 10 µM (-)gallopamil. Resting channel inhibition induced by 100 µM (-)gallopamil was 15·0 ± 1·7 % (1A-PAA/1a), 6·7 ± 1·1 % (1A-PAA/R-E/1a, P < 0·01 compared with 1A-PAA/1a), 20·1 ± 1 % (1A-PAA/2a) and 18·2 ± 1 % (1A-PAA/R-E/2a) (n 4).
Mutation Arg387Glu and 1-2a-subunit interaction differently affect recovery of Ca2+ channels from block
To elucidate the molecular basis of the different (-)gallopamil sensitivities of 1A-PAA and 1A-PAA/R-E coexpressed either with the 1a- or 2a-subunit, we analysed the drug-induced changes in IBa recovery from inactivation and block. The time courses of IBa recovery were fitted to a double-exponential function (see Methods). Under control conditions the slow component in Ca2+ channel repriming reflects recovery of Ca2+ channels from a slow or ultra-slow inactivated state (Boyett et al. 1994). (-)Gallopamil dose-dependently slowed recovery in all mutants (Fig. 2) whereas the time constant of recovery from fast inactivation was not significantly affected by the drug. The latter finding suggests that slow recovery reflects the fraction of drug-modified Ca2+ channels.
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IBa recovery of | ||
Mutation Arg387Glu did more than just slow the rate of current inactivation (see Fig. 1A). As shown in Fig. 2A and B, 1A-PAA/R-E/1a channels recovered significantly faster from inactivation (fast = 0·20 ± 0·04 s, slow = 1·9 ± 0·3 s, n = 7) compared with 1A-PAA/1a (fast = 0·34 ± 0·04 s, slow = 2·8 ± 0·4 s, n = 7, P < 0·05, Table 1). Faster IBa recovery from inactivation was accompanied by significantly faster recovery of 1A-PAA/R-E/1a channels from block by (-)gallopamil (1A-PAA/R-E/1a: slow, 100 µM = 4·4 ± 0·2 s, n = 6; 1A-PAA/1a: slow, 100 µM = 7·3 ± 0·6 s, n = 6, P < 0·05, Table 1).
Table 1. Time constants (, s) and corresponding amplitude coefficients (A) of double-exponential IBa recovery from inactivation (see Fig. 2)
| Construct | Conditions | fast |
Afast | slow |
Aslow | n |
| 1A-PAA/1A |
Control | 0·34 ± 0·04 | 0·54 ± 0·03 | 2·8 ± 0·4 | 0·33 ± 0·03 | 7 |
| 10 µM | 0·34 ± 0·06 | 0·21 ± 0·02 | 5·0 ± 0·3 | 0·57 ± 0·01 | 5 | |
| 100 µM | 0·34 * | 0·11 ± 0·02 | 7·3 ± 0·6 | 0·59 ± 0·01 | 6 | |
| 1A-PAA/R-E/1A |
Control | 0·20 ± 0·04 | 0·43 ± 0·04 | 1·9 ± 0·3 | 0·39 ± 0·04 | 7 |
| 10 µM | 0·23 ± 0·04 | 0·24 ± 0·02 | 3·3 ± 0·2 | 0·61 ± 0·02 | 4 | |
| 100 µM | 0·2 * | 0·10 ± 0·01 | 4·4 ± 0·2 | 0·75 ± 0·01 | 6 | |
| 1A-PAA/2A |
Control | 0·23 ± 0·05 | 0·073 ± 0·007 | 3·0 ± 0·2 | 0·221 ± 0·007 | 4 |
| 10 µM | 0·4 ± 0·2 | 0·09 ± 0·02 | 5·1 ± 0·4 | 0·43 ± 0·02 | 4 | |
| 100 µM | 0·23 * | 0·08 ± 0·01 | 6·3 ± 0·4 | 0·54 ± 0·01 | 4 | |
| 1A-PAA/R-E/2A |
Control | 0·24 ± 0·07 | 0·09 ± 0·01 | 2·8 ± 0·4 | 0·15 ± 0·01 | 5 |
| 10 µM | 0·28 ± 0·09 | 0·08 ± 0·01 | 3·5 ± 0·2 | 0·32 ± 0·01 | 5 | |
| 100 µM | 0·24 * | 0·071 ± 0·008 | 4·9 ± 0·2 | 0·53 ± 0·01 | 5 |
fast was fixed to fast,control.
As shown in Fig. 1A, coexpressing 1A-PAA and 1A-PAA/R-E with the 2a-subunit almost completely diminished fast IBa inactivation. This finding was confirmed by the corresponding recovery experiments. During the 3 s conditioning pulse less than 10 % of 1A-PAA/2a or 1A-PAA/R-E/2a channels accumulated in fast inactivation compared with more than 50 % of 1A-PAA/1a and 1A-PAA/R-E/1a (Fig. 2). Subsequent application of 10 or 100 µM (-)gallopamil substantially accelerated the IBa decay of all channel constructs (Fig. 3) and attenuated the slow component in IBa recovery (Fig. 2). Furthermore, drug-induced acceleration of the current decay was more prominent in 1A-PAA/2a and 1A-PAA/R-E/2a channels. As previously observed for 1A-PAA/R-E/1a, recovery of 1A-PAA/R-E/2a channels from block was more rapid than recovery of 1A-PAA/2a (Fig. 2 and Table 1).
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Normalised IBa of | ||
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DISCUSSION |
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Photoaffinity labelling experiments, radioligand binding studies and alanine scanning mutagenesis of L-type transmembrane segments IIIS6 and IVS6 suggest that the drug binding pockets for PAA, BTZ and 1,4-dihydropyridines are located near the Ca2+ channel pore (see Striessnig et al. 1998 for review). However, substitutions of inactivation determinants that have been identified in close proximity to the putative drug binding determinants on pore forming S6 segments significantly modulate sensitivity for PAA (see Hering et al. 1997) and BTZ (Berjukow et al. 1999). The latter findings suggest that those amino acids form either part of the drug-binding site or, alternatively, affect PAA and BTZ sensitivity in an indirect manner (i.e. via conformational changes modulating drug access, drug trapping or the steric orientation of the receptor determinants in the pore region, see Hering et al. 1998 for review). We have, therefore, investigated if inactivation determinants that are localised outside the putative drug-binding region have similar modulatory effects on sensitivity for the phenylalkylamine (-)gallopamil.
Here we demonstrate that an amino acid substitution (Arg387Glu) in the intracellular loop between domains I and II of the PAA-sensitive class A Ca2+ channel mutant 1A-PAA and coexpression of 1A-PAA with the 2a-subunit slow the rate of IBa inactivation and, simultaneously, reduce use-dependent Ca2+ channel block by (-)gallopamil (Fig. 1).
It is highly unlikely that Arg387 forms part of the PAA-binding site. Hence, arginine in position 387 of the 1A-PAA-subunit was mutated to the corresponding glutamate of the L-type 1C-a-subunit. Since L-type channels carry the high-affinity PAA-binding site, transfer of a L-type amino acid to 1A-PAA would be expected to increase and not, as shown here, to decrease PAA sensitivity (Fig. 1C, D and E).
It appears more likely that mutation Arg387Glu and the association of the 2a-subunit with a motif on the I-II loop known as the 'alpha interaction domain' (AID; Pragnell et al. 1994; see also Walker et al. 1998 for a second -interaction motif located on the carboxyl terminus of 1A) affect drug sensitivity indirectly by modulating channel inactivation. In other words, our data are consistent with the hypothesis that the observed effects on gallopamil sensitivity are secondary to the change in inactivation and recovery rates. It appears, therefore, likely that the different inactivation rates of naturally occurring Ca2+ channel splice variants (Zuhlke et al. 1998; Bourinet et al. 1999) affect their pharmacological properties.
Such a mechanism clearly differs from observations of Zamponi et al. (1996) indicating that the I-II loop of class A channels forms part of the piperidine receptor.
As shown in Fig. 2B, 1A-PAA/R-E/1a channels display a faster recovery from inactivation than 1A-PAA/1a (see also Table 1). 1A-PAA/R-E/1a also recovered more rapidly from block by (-)gallopamil suggesting that mutation Arg387Glu affects sensitivity for (-)gallopamil by accelerating channel unblock at rest (Table 1).
The 2a-subunit-induced changes in 1A-PAA inactivation (Fig. 1) and the consequences for use-dependent channel block by (-)gallopamil were even more dramatic (Fig. 2). Coexpression of the 2a-subunit almost completely diminished fast inactivation during a depolarising test pulse (Fig. 1A). However, the strong time-dependent block of 1A-PAA/2a channels indicates that nearly complete lack of fast inactivation does not prevent Ca2+ channel block (Fig. 3C and D). This finding is in line with the comparable resting channel inhibition by 100 µM (-)gallopamil observed for 1A-PAA/2a, 1A-PAA/R-E/2a and 1A-PAA/1a.
The time constants of 1A-PAA/2a recovery from fast and slow inactivation did not significantly differ from 1A-PAA/1a. Accordingly, drug bound 1A-PAA/2a channels recovered at nearly the same rate as the 'higher sensitive' 1A-PAA/1a channels (Fig. 2 and Table 1), and the reduced use-dependent inhibition of 1A-PAA/2a channels is, in line with the slower rate of IBa inhibition (Fig. 1E), caused by a reduced block development during membrane depolarisation.
Arg387Glu also diminished use-dependent channel block by (-)gallopamil (Fig. 1D). However, contrary to the effect of the 2a-subunit, this amino acid substitution affected not only the rate of block development but also the rate of recovery from block at rest. 'Lower PAA sensitivity' of 1A-PAA/R-E/1a (see Fig. 1) is, therefore, mainly caused by a faster channel unblock between individual pulses (Fig. 2A and B). This hypothesis fits nicely with Arg387Glu-induced changes in channel inactivation (Fig. 1B and F) that would reduce drug trapping in inactivation and facilitate recovery from block at rest (Fig. 2A and B).
Under control conditions, the effect of the Arg387Glu substitution on IBa recovery of 1A-PAA/R-E/2a was less evident than in 1A-PAA/R-E/1a channels. However, a crucial role of Arg387Glu for channel unblock was confirmed by significantly faster recovery of 1A-PAA/R-E/2a (compared with 1A-PAA/2a) in the presence of 10 and 100 µM (-)gallopamil (Fig. 2 and Table 1). In other words, reduced use-dependent block of 1A-PAA/R-E/2a (compared with 1A-PAA/2a, Fig. 1D) is also due to facilitated channel unblock between individual test pulses of a train (Table 1).
Taken together, use-dependent block was reduced either by slowing channel inactivation (caused by 2a-interaction) or by speeding recovery (mutation Arg387Glu). The additive and kinetically different effects of the 2a-subunit interaction and mutation Arg387Glu on channel block (Fig. 1D) and recovery (Fig. 2) indicate that the corresponding conformational changes in the 1-subunit are distinct and independent. Interestingly, only 1A-PAA/R-E/1a, but not 1A-PAA/2a or 1A-PAA/R-E/2a, displayed significantly different resting channel block compared with 1A-PAA/1a channels.
It is tempting to speculate that reduced use-dependent block of 1A-PAA/2a channels during a train (Fig. 1D) reflects 2a-induced changes in the steric orientation of the putative PAA binding determinants on pore forming S6-segments during a membrane depolarisation whereas Arg387Glu-induced changes in inactivation promote accelerated channel unblock. A detailed characterisation of the functional consequences of an amino acid substitution (i.e. possible modulation of fast and/or slow inactivation gating) is, therefore, a key requirement for future mutational studies directed towards the identification of drug-binding domains.
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We thank Professor H. Glossmann for continuous support of our work and Dr Stanislav Berjukow for comments on the manuscript. We also thank Drs Y. Mori and K. Imoto for the gift of the 1A cDNA, Dr A. Schwartz for providing the 2/ cDNA, Dr L. Birnbaumer for providing the 2a cDNA and Dr Traut (Knoll AG, Ludwigshafen, Germany) for providing (-)gallopamil. This work was supported by grants from the Fonds zur Förderung der Wissenschaftlichen Forschung P12649 (S.H.), a grant from the Else Kröner Fresenius Stiftung (S.H.), and a grant from the Österreichische Nationalbank (S.H.), and is part of the thesis of S.S.
Corresponding author
S. Hering: Institut für Biochemische Pharmakologie, Peter-Mayr-Straße 1, A-6020 Innsbruck, Austria.
Email: steffen.hering{at}uibk.ac.at
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) or in the presence of 10 µM (
). Bars represent the means ± S.E.M. (n = 5-8). * Significantly different from IBa block of 
Significantly different from 
, 
, 
, 
, 
) and 
) in control. Curves are drawn according to the equation I/Imax = Iss + (1 - Iss)/(1 + exp[(V - V0·5)/k]) (see Methods) with V0·5 = -11·3 ± 0·5 mV, k = 7·4 ± 0·4 mV, Iss = 0·03 ± 0·01, n = 5 for