Mechanism of Dihydropyridine Interaction with Critical Binding Residues of L-type Ca2+ Channel alpha 1 Subunits*

Edwin Wappl, Jörg Mitterdorfer, Hartmut Glossmann, and Jörg StriessnigDagger

From the Institut für Biochemische Pharmakologie, Peter-Maystrasse 1, A-6020 Innsbruck, Austria

Received for publication, November 8, 2000, and in revised form, January 12, 2001



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We investigated the mechanism of interaction of individual L-type channel amino acid residues with dihydropyridines within a dihydropyridine-sensitive alpha 1A subunit (alpha 1ADHP). Mutation of individual residues in repeat III and expression in Xenopus oocytes revealed that Thr1393 is not required for dihydropyridine interaction but that bulky side chains (tyrosine, phenylalanine) in this position sterically inhibit dihydropyridine coordination. In position 1397 a side chain carbonyl group was required for high antagonist sensitivity. Agonist function required the complete amide group of a glutamine residue. Val1516 and Met1512 side chains were required for agonist (Val1516) and antagonist (Val1516, Met1512) sensitivity. Replacement of Ile1504 and Ile1507 by alpha 1A phenylalanines was tolerated. Substitution of Thr1393 by phenylalanine or Val1516 by alanine introduced voltage dependence of antagonist action into alpha 1ADHP, suggesting that these residues form part of a mechanism mediating voltage dependence of dihydropyridine sensitivity. Our data provide important insight into dihydropyridine binding to alpha 1ADHP which could facilitate the development of alpha 1A-selective modulators. By modulating P/Q-type Ca2+ channels such drugs could serve as new anti-migraine therapeutics.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The membrane depolarization-dependent opening of voltage-gated Ca2+ channels effectively modulates Ca2+ influx into electrically excitable cells. These channels represent key elements in Ca2+ signaling, controlling neurotransmitter release, synaptic plasticity, muscle contraction, and pacemaker functions. So far selective nonpeptide Ca2+ channel modulators have only been developed for L-type Ca2+ channels which are expressed primarily in the cardiovascular system but also in neuronal and neuroendocrine cells (1, 2). Other Ca2+ channel types, such as N, P, Q, and R-type Ca2+ channels, play a prominent role for fast neurotransmitter release in neurons (1), but they are insensitive to L-type Ca2+ channel modulators. 1,4-Dihydropyridines (DHPs),1 such as isradipine or amlodipine, are very well characterized L-type Ca2+ channel blockers ("antagonists") that are also used to treat cardiovascular diseases. In contrast, DHP Ca2+ channel activators ("agonists," e.g. BayK8644) stimulate Ca2+ currents through L-type channels. DHPs do not modulate ion currents through, e.g. P- or Q-type channels.

This difference in DHP sensitivity is the result of different alpha 1 subunit isoforms which, together with accessory subunits such as alpha 2-delta and beta , form the Ca2+ channel complexes. L-type Ca2+ channels are formed, e.g. by DHP-sensitive alpha 1C or alpha 1S subunits, whereas P- and Q-type Ca2+ channels contain DHP-insensitive alpha 1A subunits.

Individual amino acid residues that form the DHP binding pocket in L-type alpha 1 subunits have recently been identified (for review, see Refs. 3-5). Most interestingly, some but not all of these residues are conserved in non-DHP-sensitive subunits, such as alpha 1A and alpha 1E. These subunits are rendered fully DHP agonist- and antagonist-sensitive after introduction of the remaining nonconserved L-type residues forming the binding pocket (yielding, e.g. DHP-sensitive alpha 1A subunits, alpha 1ADHP (6, 7)).

Although site-directed mutagenesis has been used to identify residues important for DHP interaction, the exact role of some of these residues in antagonist and agonist interaction has not been completely studied. In addition, for some of these L-type residues it is still unclear if their introduction into alpha 1A creates additional interaction sites for the DHP molecule or removes an inhibitory effect of the corresponding alpha 1A residue. A more detailed insight into the molecular mechanisms of DHP binding to its recombinant drug binding domain in alpha 1ADHP could provide important structural information for developing small molecules with affinity for alpha 1A subunits. Such drugs could be useful therapeutics for the treatment of alpha 1A-associated disorders such as migraine (8).

To address this question we used site-directed mutagenesis to study further the mechanism of DHP interaction with L-type residues in repeat III of a DHP-sensitive alpha 1A subunit (alpha 1ADHP). By carrying out these studies within the alpha 1A background our results can be applied to future studies aimed at the development of alpha 1A-selective Ca2+ channel modulators.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction of Mutant alpha 1 cDNAs-- The construction of alpha 1ADHP and M1512V (alpha 1ADHPi) was described previously (6). Mutations were introduced into alpha 1ADHP cDNA containing silent restriction sites (indicated by asterisks) generated by polymerase chain reaction (PCR) in previous cloning steps (9). Mutant alpha 1 cDNAs were created by PCR using the "gene SOEing" technique (10) employing proofreading Pfu polymerase (Stratagene).

Mutations of Thr1393 and Gln1397 (alpha 1A numbering (11); corresponding to positions 1039 and 1043 in alpha 1C-II, respectively (12)) were introduced by PCR using appropriately mutated SOE-PCR primers into a SfiI-SalI* cassette (nucleotides 4297-4744, alpha 1A numbering) of an XhoI-ClaI* fragment (nucleotides 1689-4925, domains II and III) of alpha 1ADHP subcloned into pBluescript SK+ (Stratagene). The mutations were reintroduced through an NheI-ClaI* fragment (nucleotides 3543-4925, domain III) into alpha 1ADHP in PNKS2 (provided by O. Pongs, ZFMNB, Hamburg, Germany), thus yielding the following alpha 1ADHP mutants: T1393A, T1393Y, T1393F, T1393S, Q1397A, Q1397M, Q1397E, and Q1397N. Mutations in transmembrane segment IIIS6 were introduced by PCR into an SalI*-ClaI* cassette (nucleotides 4744-4925) of alpha 1ADHP in PNKS2, thus yielding the following alpha 1ADHP mutants: Y1503A, I1504A, I1504F, I1507A, I1507F, P1508A, M1512A, and V1516A, corresponding to positions 1152, 1153, 1156, 1157, 1161, and 1165 in alpha 1C-II, respectively.

Mutagenic primers contained silent point mutations to introduce or eliminate restriction endonuclease sites for verification of the desired mutations. Fragments amplified by PCR were sequenced entirely to confirm sequence integrity.

Expression of alpha 1 Mutants in Xenopus laevis Oocytes-- Capped run-off poly(A+) cRNA transcripts from XbaI-linearized cDNA templates were synthesized according to Krieg and Melton (13). alpha 1 cRNA was coinjected with beta 1a (14) and alpha 2delta (15) subunit cRNAs into stage V-VI oocytes from X. laevis.

Electrophysiological Measurements-- 1-6 days after cRNA injection inward barium currents (IBa) through voltage-gated Ca2+ channels were measured at room temperature using the two-microelectrode voltage-clamp technique as described previously (9). To quantifiy endogenous IBa X. laevis oocytes injected only with beta 1a and alpha 2delta were analyzed in parallel. Only oocytes expressing peak IBa through recombinant Ca2+ channels at least three times as high (usually >100 nA) as the highest endogenous currents were included into analysis. Data analysis and acquisition were performed by using the pClamp software package (version 6.0, Axon Instruments). Leakage correction was performed by adjusting the current traces by a factor calculated from the difference between the leak at -80 mV and -90 mV, respectively.

The extracellular solution contained 40 mM Ba(OH)2, 50 mM NaOH, 2 mM CsOH, and 5 mM HEPES (pH adjusted to 7.4 with methanesulfonic acid). The voltage recording and current-injecting microelectrodes were filled with 2.8 M CsCl, 0.2 M CsOH, 10 mM HEPES, and 10 mM EGTA (adjusted to pH 7.4 with HCl) and had resistances of 0.7-6 megohms.

Modulation of peak IBa was measured from the indicated standard holding potentials to a test pulse corresponding to the peak potential (IBa block by DHP antagonists) or 10 mV positive to the peak potential of the current-voltage relations (IBa stimulation by DHP agonist and FPL64176, respectively). Standard pulse frequency was 0.017 or 0.034 Hz to assess channel modulation. Isradipine was employed at a concentration (10 µM) causing near complete channel block in oocytes at negative holding potentials (6).

The time course of peak current inhibition by isradipine was estimated by fitting the peak currents of successive episodes to a monoexponential function (IBa = A*exp(-t/tau ) + C).

Because of slow recovery from inactivation, steady-state inactivation was quantified from the decline of peak IBa after switching from a holding potential of -120 mV to more positive holding potentials allowing equilibrium IBa to be reached. For mutant V1516A, which showed relatively little inactivation at -80 mV, -100 mV was used as reference potential for the resting state.

Reagents-- (±)-Isradipine (PN200-110) was from Sandoz AG (Basel, Switzerland), (±)-BayK8644 was from Bayer AG (Wuppertal, Germany), and FPL64176 was from Fisons Pharmaceuticals (Leicestershire, U. K.). Drug-containing solutions were freshly prepared from 10 mM stock solutions (in dimethyl sulfoxide) and applied at the same flow rate as control solution.

Statistics-- Data are given as the mean ± S.D. Statistical significance was calculated using the unpaired Student's t test, employing OriginR (Microcal, version 6.0).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We and others have shown recently that alpha 1A (alpha 1ADHP, Fig. 1A) and alpha 1E subunits become highly DHP-sensitive by mutation of 8-9 amino acids to residues present in L-type alpha 1 subunits (6, 7, 16). In this study we used alpha 1ADHP as a suitable model to study further the mechanism by which amino acid residues in transmembrane segments IIIS5 and IIIS6 participate in DHP interaction. The alpha 1A structural background should allow application of the results for alpha 1A drug development.


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Fig. 1.   Effects of single mutations in IIIS5 on DHP sensitivity. A, schematic representation of alpha 1ADHP. L-type residues (black circles) are given in single-letter code. B, amino acid sequence alignment of alpha 1C, alpha 1A, alpha 1ADHP, and alpha 1ADHP mutants. Nonconserved L-type channel amino acids (black boxes) and substituted amino acids in mutants of alpha 1ADHP (gray boxes) are highlighted. C, isradipine (10 µM) inhibition and BayK8644 (10 µM) stimulation of peak IBa through mutant Ca2+ channels expressed in X. laevis oocytes. Drug effects were determined at different holding potentials (black bars, -80 mV; white bars, -100 mV). Data are presented as the means ± S.D. from the indicated number of experiments; statistically significant differences from alpha 1ADHP (-80 mV) are indicated: *, p < 0.05; **, p < 0.01.

Role of Thr1393 in Transmembrane Helix IIIS5-- Previous studies have shown that replacement of the IIIS5 residues Thr1393 or Gln1397 (numbering according to alpha 1ADHP, see also Fig. 4) in DHP-sensitive alpha 1A chimeras (17) or alpha 1C (18) by the corresponding alpha 1A residues (T1393Y, Q1397M) substantially decreased DHP sensitivity. It is unclear if this is because of a steric interference introduced by the corresponding tyrosine or methionine side chain, respectively, or the result of the removal of the appropriate pharmacophores. To address this question we replaced Thr1393 or Gln1397 in alpha 1ADHP by a series of other residues and tested the consequences of these mutations for modulation of IBa by the DHP Ca2+ channel blocker isradipine and activator BayK8644 after expression in Xenopus oocytes. In agreement with previous results, 10 µM isradipine inhibited 72.1 ± 6.9% (n = 4, Fig. 1C) of IBa through alpha 1ADHP elicited from a holding potential of -80 mV. 10 µM BayK8644 stimulated IBa by 4.3 ± 1.2-fold (n = 4). Sensitivity was abolished after mutation of Thr1393 to tyrosine (mutant T1393Y: 4 ± 8.3% inhibition, n = 5; 1.4 ± 0.7-fold stimulation, n = 7). T1393Y was also insensitive to stimulation by the non-DHP agonist FPL64176 (1.04 ± 0.1 fold stimulation, n = 3). Fig. 1C shows that reduction of the side chain size by mutation from threonine to serine (mutant T1393S: 59.2 ± 16.3% inhibition, n = 3; 3.6 ± 0.4-fold stimulation, n = 5; p > 0.05) or alanine (mutant T1393A: 59.5 ± 10.3% inhibition, n = 5; 5.6 ± 2.8-fold stimulation, n = 4, p > 0.05) not only preserved full DHP antagonist sensitivity but also supported stimulation by BayK8644. These results show that the side chain of Thr1393 is not essential for DHP modulation. Instead, the bulky hydroxyphenyl moiety of tyrosine must prevent DHP interaction (19).

To determine whether DHP antagonist sensitivity was affected by membrane voltage, inhibition by isradipine was measured also at -100 mV holding potential at which a much smaller fraction of channels underwent steady-state inactivation than at -80 mV (Table I). As for alpha 1ADHP (Fig. 2; 20), DHP sensitivity was not affected by membrane voltage in T1393A (-100 mV: 62.1 ± 10, 5% inhibition, n = 4) and T1393Y (Fig. 2;). No significant antagonist sensitivity of T1393Y was recovered at holding potentials causing 70% steady-state inactivation (12.0 ± 13.2%, n = 3; Fig. 2). Steady-state inactivation properties of mutant T1393Y were similar to alpha 1ADHP (Table I). This rules out decreased antagonist sensitivity of T1393Y being caused by changes in channel inactivation properties.

                              
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Table I
Steady-state inactivation of alpha 1ADHP and alpha 1ADHP mutants
Steady-state inactivation was determined at -80 and -100 mV as described under "Experimental Procedures." Data are given as the means ± S.D. (n = 3-10). Statistical significant differences from alpha 1ADHP are indicated. *, p < 0.05; **, p < 0.01; ND, not determined.


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Fig. 2.   Effects of inactivation on DHP antagonist sensitivity. A, representative IBa traces illustrate the sensitivity of alpha 1ADHP, T1393Y, and T1393F to inhibition by 10 µM isradipine at different holding potentials. One typical recording out of at least three experiments is shown. IBa was elicited by depolarizations to potentials corresponding to the peak of the current-voltage relations. ISR, isradipine; Co, control. 70% channel inactivation was achieved at the following holding potentials: alpha 1ADHP, -65 mV; T1393Y, -61 mV; T1393F, -70 mV. 50% channel inactivation was achieved at -74 mV (T1393F). Cells: alpha 1ADHP: -120 mV, e0724029; 70% inactivation, e0309024; T1393Y: -100 mV, e9317029; 70% inactivation, e0218051; T1393F: -100 mV, e9818020; -80 mV, e9506033; 50% inactivation, e0309036; 70% inactivation, e0309031. B, inhibition of peak IBa by 10 µM isradipine through alpha 1ADHP and the indicated mutants at different holding potentials. 70% channel inactivation was achieved at the following holding potentials: alpha 1ADHP, -65-74 mV; T1393Y, -61-70 mV; T1393F, -70-77 mV; V1516A, -50-55 mV. 50% channel inactivation was achieved at the following holding potentials: T1393F, -74-80 mV; V1516A, -50-62 mV. Data are presented as the mean ± S.D. from the indicated number of experiments; a statistically significant difference from alpha 1ADHP at the same holding potential (or at -80 mV) is indicated: *, p < 0.05; **, p < 0.01.

Next we investigated whether the hydroxyl group of the tyrosine side chain contributes to the reduction of DHP sensitivity in T1393Y by creating mutant T1393F. As illustrated in Fig. 2, the isradipine sensitivity of peak IBa through T1393F was reduced dramatically at -100 mV holding potential (8.8 ± 5.0% inhibition, n = 3), and stimulation by BayK8644 was negligible (1.34 ± 0.28-fold stimulation, n = 3, Fig. 1). However, isradipine increased IBa decay during depolarization causing 42 ± 20% (n = 3) inhibition at the end of the 350-ms test pulse (Fig. 2A). This indicated that the drug still interacts with depolarized channels. The increase in IBa decay may be caused by preferential interaction with or promotion of inactivated channel states (20) and prompted us to study DHP block at more depolarized holding potentials. Unlike T1393Y, DHP antagonist sensitivity of T1393F increased substantially at more positive holding potentials, and inhibition of peak IBa was recovered completely at potentials causing 70% steady-state inactivation (Fig. 2). Interestingly, development of block was slowed compared with alpha 1ADHP (T1393F: tau  = 4.51 ± 1.38 min, n = 6; alpha 1ADHP: tau  = 2.28 ± 0.26 min, n = 4, p < 0.05).

No evidence for a similar voltage-dependent action was found for BayK8644 (Fig. 1C). Taken together our experiments show that the tyrosine's phenyl ring alone is sufficient to decrease alpha 1ADHP antagonist sensitivity at negative potentials but that the additional hydroxyl group is required to maintain this effect at more positive voltages.

Role of Gln1397 in IIIS5-- Previous experiments have shown that mutation of IIIS5 Gln1397 to the corresponding methionine (17, 18) reduces DHP sensitivity. This was also observed after introduction of this mutation (Q1393M) into alpha 1ADHP (Fig. 1C). Antagonist sensitivity was decreased, and simulation by the agonists FPL64176 (0.95 ± 0.13-fold stimulation, n = 3) and BayK8644 (Fig. 1C) was abolished. In contrast to Thr1393, introduction of an alanine, resulting in mutant Q1397A, did not support full DHP antagonist (29.6 ± 13.5% inhibition, n = 7) and agonist (1.4 ± 0.15-fold stimulation, n = 5) sensitivity (Fig. 1C). This indicates that the side chain of Gln1397 participates directly in DHP binding. Replacement of glutamine by asparagine, which shortens the side chain by one methylene group, conferred full antagonist sensitivity (68.8 ± 9.8%, n = 5, p > 0.05) but significantly reduced stimulation by the agonist (1.8 ± 0.66-fold, n = 5). A similar finding was obtained for mutant Q1397E (63.5 ± 15% inhibition, n = 5; 1.8 ± 0.36-fold stimulation, n = 4) and Q1397D (17) in which the side chain NH2 groups are replaced by OH groups. Taken together these results confirm our previous hypothesis that the carbonyl group (present in the amide and carboxyl side chains) in position 1397 is sufficient for DHP antagonist sensitivity, whereas full agonist action requires the complete amide moiety. Our data show that the latter must even be located in an appropriate distance from the IIIS5 backbone because only glutamine but not asparagine supports full activity.

Role of IIIS6 Residues-- Previous radioligand binding (21) or functional (22) studies have shown that Tyr1503, Ile1504, Ile1507, and Met1512 are important determinants of isradipine affinity in transmembrane helix IIIS6. However, the contribution of Tyr1503, Ile1504, and Ile1507 have not yet been determined within the alpha 1ADHP background. As shown in Fig. 3B, mutation of these residues to alanine did not decrease antagonist sensitivity (-80 mV). This is consistent with previous results obtained with mutated alpha 1C subunits, in which only 2.5-5-fold lower affinities at -80 mV were reported for these single mutations in functional experiments (22). As in alpha 1C (22), mutation Y1503A, but not I1504A and I1507A, abolished agonist stimulation of peak current (Fig. 3B). The activating effect of BayK8644 was converted into an inhibitory one, evident as inhibition of 42 ± 8.4% (n = 3) of IBa at the end of the 350-ms test pulse (Fig. 3C). This demonstrates that the mutation does not block DHP interaction with the channel but prevents the agonist from stabilizing open channel conformations. Because BayK8644 is employed as the racemic mixture of an agonist ((-)-enantiomer) and a weak antagonist ((+)-enantiomer), IBa inhibition most likely reflects channel block by the antagonistic enantiomer in the absence of stimulation by the agonist. This is supported further by the finding of the complete lack of IBa modulation by the optically pure agonist FPL64176 (0.97 ± 0.12-fold stimulation, n = 3).


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Fig. 3.   Effects of DHPs on IBa through IIIS6 mutant channels. A, amino acid sequence alignment of alpha 1C, alpha 1A, alpha 1ADHP, and alpha 1ADHP mutants. Nonconserved L-type channel amino acids (black boxes) and substituted amino acids in mutants of alpha 1ADHP (gray boxes) are highlighted. B, isradipine (10 µM) inhibition and BayK8644 (10 µM) stimulation of peak IBa through mutant Ca2+ channels expressed in X. laevis oocytes at -80 mV holding potential. Data are given as the means ± S.D. from the indicated number of experiments; statistically significant differences from alpha 1ADHP are indicated: *, p < 0.05; **, p < 0.01. C, representative IBa traces; one of at least three experiments is illustrated. IBa were evoked by depolarizations to potentials 10 mV negative of the peak of the current-voltage relations from a holding potential of -80 mV. Co, control current before drug application; FPL, 10 µM FPL64176 present; BayK, 10 µM BayK8644 present. Cells: alpha 1ADHP: j6n09c53; I1507F: BayK, e9o01028; FPL, e0115034; Y1503A: e9d11045.

To address the question of whether DHP sensitivity is still maintained after back-mutation of L-type residues in positions 1504 and 1507 to the corresponding alpha 1A phenylalanines, we constructed mutants I1504F and I1507F. Both mutants showed only minor and nonsignificant decreases in isradipine inhibition compared with alpha 1ADHP. This demonstrates that the DHP sensitivity of alpha 1A can be achieved by introduction of an even lower number of L-type residues into alpha 1ADHP than described previously. Both mutants were efficiently stimulated by 10 µM BayK8644. In the case of I1507F, stimulation was even more pronounced than for alpha 1ADHP (15.6 ± 7.6-fold, n = 7). Such an enhancement was also found for stimulation by FPL64176, although not on the level of statistical significance (I1507F: 4.1 ± 1.16-fold, n = 4, Fig. 3C; alpha 1A-DHP: 2.8 ± 0.84-fold, n = 3 (6)).

We also analyzed the requirement of the Met1512 side chain for agonist action. In agreement with earlier studies (21) mutation M1512A reduced isradipine sensitivity (Fig. 3B), suggesting that the Met1512 side chain interacts directly with the DHP antagonist. This side chain preferentially mediated antagonist effects because stimulation by BayK8644 was not affected by the mutation (5.5 ± 2.6-fold stimulation, n = 6). This is in contrast to our previous observation in which substitution of this residue by a valine reduced both agonist and antagonist sensitivity (Fig. 3B (6)).

Other residues in IIIS6 which may affect drug modulation by DHPs are Pro1508 and Val1516. In position 1508 both alanine and proline support high affinity DHP antagonist interaction in alpha 1C (23) and alpha 1ADHP (Fig. 3B). However, mutation of Pro1508 to alanine may cause mutation-induced conformational changes of the IIIS6 helix (24) which could affect modulation by agonists. This possibility has not yet been investigated. We found no evidence for such an effect in P1508A (Fig. 3B). Val1516, which is conserved in L- and non-L-type Ca2+ channel alpha 1 subunits, was recently found to contribute to Ca2+ channel block by phenylalkylamines (25) and (+)-cis-diltiazem (26), but its contribution to DHP sensitivity is unknown. Figs. 2 and 3 illustrate that mutation V1516A decreased stimulation by BayK8644 and inhibition by isradipine at a -80 mV holding potential. As for T1393F, V1516A increased IBa decay during the test pulse. At -80 mV peak IBa was reduced by 21 ± 12% (n = 5), but IBa at the end of the 350-ms test pulse was reduced by 55 ± 15% (n = 5). This mutation also introduced voltage dependence for isradipine block, as decreased DHP sensitivity was not observed at holding potentials where 70% of the channels were inactivated (Fig. 2). In contrast to T1393F, no slowing of isradipine block development was evident (tau  = 1.53 ± 0.96 min, n = 3).

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have studied the contribution of residues previously found to mediate sensitivity of L-type Ca2+ channel alpha 1 subunits to DHP Ca2+ channel blockers and activators. Our data (summarized in Fig. 4) provide further structural insight into the differential affinity of alpha 1A and L-type alpha 1 subunits for DHP Ca2+ channel modulators.


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Fig. 4.   Summary of the role of individual IIIS5 and IIIS6 amino acids analyzed. The alpha 1ADHP sequence in the (putative) IIIS5 and IIIS6 alpha -helices is illustrated.

Decreased DHP sensitivity after the introduction of bulky tyrosine or phenylalanine residues into position 1393 of alpha 1A (T1393Y, T1393F) is not caused by the removal of a critical binding contact. Instead, the bulky side chains sterically prevent high affinity DHP interaction. In T1393F, i.e. in the absence of the tyrosine hydroxyl group, obstruction of DHP antagonist interaction is only found for resting channels but is nearly absent when channels are inactivated. It is tempting to speculate that the hydroxyl function stabilizes a certain orientation of the tyrosine side chain which interferes with DHP antagonist coordination independent of channel conformation. This may be accomplished, e.g. by forming a hydrogen bond with other residues. From a working model of the DHP binding domain (19) based on the three-dimensional coordinates of the kcsA potassium channel (24) the carbonyl oxygen of the IIIS6 Met1511 would be the most likely candidate for such hydrogen bonding (not shown). Removal of the hydroxyl group (in mutant T1393F) still prevents high DHP antagonist sensitivity of resting channels but restores sensitivity when the channels inactivate. We propose that hydrogen bonding does not occur without the hydroxyl group, providing the phenyl group with more rotational flexibility such that DHP antagonist binding to inactivated states is possible. This flexibility may delay, but not prevent, DHP association to the channel. Such a model can explain the observed slower onset of DHP block of IBa through T1393F compared with alpha 1ADHP.

We present evidence that, in contrast to Thr1393, the Glu1397 side chain is involved directly in DHP binding. Maximal DHP antagonist sensitivity critically depends on the presence of a side chain carbonyl group, a potential hydrogen bond acceptor, whereas DHP agonist action requires an intact amide function spaced at a defined distance (glutamine amide) from the IIIS5 protein backbone.

Introduction of nine L-type channel amino acids into alpha 1A renders the resulting DHP-sensitive construct (alpha 1ADHP) highly sensitive to DHPs. Although alpha 1ADHP retains most of the hallmarks of DHP interaction with L-type Ca2+ channel alpha 1A subunits (such as high affinity, stereoselectivity, and allosteric modulation by non-DHP Ca2+ antagonists (19)), the typical voltage dependence of DHP block is absent (20). This has also been described previously for a DHP-sensitive alpha 1E subunit construct (27). We show that the change of only a single amino acid residue can introduce considerable voltage dependence of the DHP block into alpha 1ADHP. This is an important observation because the molecular mechanism for voltage-dependent Ca2+ channel block by DHPs is not well understood. Our experiments clearly demonstrate that first, alterations of single amino acid side chains affect voltage-dependent DHP block. In previous studies the voltage dependence of alpha 1C subunits has been associated with sequence divergence of multiple residues within larger sequence stretches (comprising transmembrane segment IS6 (27, 28)). Second, we found that single mutations within two distinct but adjacent transmembrane segments of the DHP binding domain can modify the voltage dependence of DHP inhibition of alpha 1ADHP by apparently different mechanisms. The addition of a bulky phenylalanine in position 1393 mainly decreases DHP antagonist sensitivity for resting channels. This steric hindrance decreases when the channel assumes the inactivated conformation (see above). A different mechanism seems to apply for mutation V1516A. In this case, DHP sensitivity also decreases mainly for resting channels after removal of the valine side chain by mutation to alanine. This suggests that the valine side chain is essential for DHP action in the resting channel conformation but is not important once the channel inactivates. The relative contribution of individual residues for DHP binding must therefore change upon depolarization. We cannot distinguish whether the Val1516 side chain interacts directly with the DHP molecule in the resting state or contributes indirectly to a hydrophobic stabilization of the binding domain which is disrupted by its mutation to alanine. Taken together, our experiments identify two basic mechanisms that explain voltage-dependent changes in DHP sensitivity, at least in alpha 1ADHP Ca2+ channels: gating-induced molecular rearrangements within the DHP binding domain can alter DHP antagonist affinity either by changing the extent of steric hindrance by a particular residue (such as in T1393F) or by providing (or stabilizing) suitable attachment sites for the drug (such as in V1516A).

Our results contribute important information for the development of P/Q-type Ca2+ channel-selective modulators. DHP derivatives would represent ideal lead compounds for the development of alpha 1A Ca2+ channel modulators. Investigation of the DHP binding domain within the alpha 1A sequence background of alpha 1ADHP not only reveals information about the molecular mechanism of DHP interaction with L-type Ca2+ channel alpha 1 subunits in general, but also provides structural hints for novel DHP analogs that could possess considerable affinity for alpha 1A subunits and therefore represent lead compounds for further drug development.

We found that in the alpha 1A sequence environment the two L-type isoleucine residues in IIIS6 (Ile1504, Ile1507) are not important for DHP interaction and can be replaced by the respective alpha 1A phenylalanines. Therefore transfer of high DHP sensitivity to alpha 1A requires the introduction of even less than 9 L-type residues as reported for alpha 1ADHP (6, 7). From one of our previous studies with alpha 1A chimeras (9) it is evident that DHP antagonist sensitivity only very weakly depends on the four L-type residues in repeat IV (Fig. 1A). Therefore, DHP interaction with the remaining three L-type channel amino acid residues in IIIS5 and IIIS6 (Thr1393, Gln1397, and Met1512) is crucial for DHP sensitivity of alpha 1ADHP. The strongest structural determinant abolishing DHP sensitivity in alpha 1A is the tyrosine in position Thr1393 which, according to our data, sterically prevents DHP binding. In contrast, there is no evidence for such an effect for alpha 1A residues introduced into positions 1397 and 1512. Instead, these fail to provide suitable attachment sites for the DHP molecule thus leading to a measurable but limited sensitivity decrease. It is therefore likely that drug molecules (e.g. DHP derivatives) that escape the steric hindrance of Tyr1393 and do not depend on binding interaction with Met1512 and Gln1397 must have considerable Ca2+ channel blocking effects for alpha 1A subunits. Smaller and more hydrophobic compounds may fulfill these requirements.

Together with the results from earlier studies (9, 17, 18, 22, 29) our data show that the structural requirements for effective stimulation by DHP agonists of IBa through alpha 1ADHP are more complex than for antagonists. This is based on the observation that introduction of the alpha 1A sequence into several L-type positions of alpha 1ADHP in repeats III (1393, 1397, 1512) and IVS6 (9) results in a complete loss of agonist sensitivity to 10 µM concentrations of BayK8644 and FPL64176. Most of these mutations and mutation Y1503A illustrate that DHP agonist effects can be removed completely in mutants still displaying antagonist sensitivity (e.g. Q1397M, Y1503A, M1512V; Fig. 3). In radioligand binding studies DHP agonists and antagonists bind to their binding domain in an apparently competitive manner. It is therefore likely that such mutations still allow DHP agonist binding to the channel but are unable to stabilize or prevent stabilization of open channel conformations by the bound agonist. A similar conclusion was reached after mutation of a pore-loop serine (position 1466 in alpha 1ADHP), which prevents agonist effects but appears still to mediate antagonist sensitivity (5).

In summary, our data provide important insight into DHP interaction with their binding domain within the structural background of alpha 1A. This molecular information provides clues for the development of alpha 1A-selective Ca2+ channel blockers.

    ACKNOWLEDGEMENTS

We thank Drs. T. Langer (Institute of Pharmaceutical Sciences), M. Grabner, S. Hering, and S. Berjukow for helpful discussions, Drs. M. Sinnegger and R. Kraus for reading the manuscript, and E. Emberger and D. Kandler for expert technical assistance.

    FOOTNOTES

* This work was supported by Austrian Science Fund Grants P12641 and P14820 (to J. S.) and P12689 (to H. G.), by the Österreichische Nationalbank, the Dr. Legerlotz Foundation, and European Community Research Training Network Grant HPRN-CT-2000 00082 (to J. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 43-512-507-3164; Fax: 43-512-588-627; E-mail: joerg.striessnig@uibk. ac.at.

Published, JBC Papers in Press, January 19, 2001, DOI 10.1074/jbc.M010164200

    ABBREVIATIONS

The abbreviations used are: DHP(s), 1,4-dihydropyridine(s); PCR, polymerase chain reaction; IBa, inward barium current(s).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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