Molecular Mechanism of Diltiazem Interaction with L-type Ca2+ Channels
1998; Elsevier BV; Volume: 273; Issue: 42 Linguagem: Inglês
10.1074/jbc.273.42.27205
ISSN1083-351X
AutoresRichard L. Kraus, Steffen Hering, Manfred Grabner, Dominique Ostler, Jörg Striessnig,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoBenzothiazepine Ca2+antagonists (such as (+)-cis-diltiazem) interact with transmembrane segments IIIS6 and IVS6 in the α1 subunit of L-type Ca2+ channels. We investigated the contribution of individual IIIS6 amino acid residues for diltiazem sensitivity by employing alanine scanning mutagenesis in a benzothiazepine-sensitive α1 subunit chimera (ALDIL) expressed in Xenopus laevis oocytes.The most dramatic decrease of block by 100 μm diltiazem (ALDIL 45 ± 4.8% inhibition) during trains of 100-ms pulses (0.1 Hz, −80 mV holding potential) was found after mutation of adjacent IIIS6 residues Phe1164(21 ± 3%) and Val1165 (8.5 ± 1.4%). Diltiazem delayed current recovery by promoting a slowly recovering current component. This effect was similar in ALDIL and F1164A but largely prevented in V1165A. Both mutations slowed inactivation kinetics during a pulse. The reduced diltiazem block can therefore be explained by slowing of inactivation kinetics (F1164A and V1165A) and accelerated recovery from drug block (V1165A). The bulkier diltiazem derivative benziazem still efficiently blocked V1165A.From these functional and from additional radioligand binding studies with the dihydropyridine (+)-[3H]isradipine we propose a model in which Val1165 controls dissociation of the bound diltiazem molecule, and where bulky substituents on the basic nitrogen of diltiazem protrude toward the adjacent dihydropyridine binding domain. Benzothiazepine Ca2+antagonists (such as (+)-cis-diltiazem) interact with transmembrane segments IIIS6 and IVS6 in the α1 subunit of L-type Ca2+ channels. We investigated the contribution of individual IIIS6 amino acid residues for diltiazem sensitivity by employing alanine scanning mutagenesis in a benzothiazepine-sensitive α1 subunit chimera (ALDIL) expressed in Xenopus laevis oocytes. The most dramatic decrease of block by 100 μm diltiazem (ALDIL 45 ± 4.8% inhibition) during trains of 100-ms pulses (0.1 Hz, −80 mV holding potential) was found after mutation of adjacent IIIS6 residues Phe1164(21 ± 3%) and Val1165 (8.5 ± 1.4%). Diltiazem delayed current recovery by promoting a slowly recovering current component. This effect was similar in ALDIL and F1164A but largely prevented in V1165A. Both mutations slowed inactivation kinetics during a pulse. The reduced diltiazem block can therefore be explained by slowing of inactivation kinetics (F1164A and V1165A) and accelerated recovery from drug block (V1165A). The bulkier diltiazem derivative benziazem still efficiently blocked V1165A. From these functional and from additional radioligand binding studies with the dihydropyridine (+)-[3H]isradipine we propose a model in which Val1165 controls dissociation of the bound diltiazem molecule, and where bulky substituents on the basic nitrogen of diltiazem protrude toward the adjacent dihydropyridine binding domain. (+)-cis-diltiazem (3R,4S)-cis-1-[2-[3-(benzoylamino)propyl]amino-ethyl]-1,3,4,5-tetrahydro-3-hydroxy-4-(4-methoxyphenyl)-6-(trifluoromethyl)-2H-1-benzazepine-2-one benzothiazepine dihydropyridine (3R,4S)-cis-1-[2-[[3-[[3-[4,4-difluoro-3a,4-dihydro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-yl]proprionyl]amino]propyl]amino]ethyl]-1,3,4,5-tetrahydro-3-hydroxy-4-(4-methoxyphenyl)-6-(trifluoromethyl)-2H-1-benzazepine-2-one barium inward current phenylalkylamine. The activity of voltage-gated L-type Ca2+ channels in muscle, endocrine and neuronal cells is blocked by different chemical classes of drugs, termed Ca2+ antagonists. Dihydropyridines (nifedipine, isradipine), phenylalkylamines (verapamil, desmethoxyverapamil) and benzothiazepines ((+)-cis-diltiazem; diltiazem)1 stereoselectively interact with high affinity binding domains on the so-called α1 subunit of the L-type Ca2+ channel complex and thereby cause channel block (1Glossmann H. Striessnig J. Rev. Physiol. Biochem. Pharmacol. 1990; 114: 1-105Crossref PubMed Google Scholar). Recent biochemical studies, employing photoaffinity labeling, site-directed mutagensis, and chimeric α1 subunit constructs revealed that high affinity binding determinants for these drugs are located close to pore-forming regions of L-type Ca2+ channel α1 subunits. Important binding determinants for DHPs and PAAs were identified in transmembrane segments IIIS5, IIIS6, and IVS6 (for review, see Refs. 2Hockerman G.H. Peterson B.Z. Johnson B.D. Catterall W.A. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 361-396Crossref PubMed Google Scholar and 3Striessnig J. Grabner M. Mitterdorfer J. Hering S. Sinnegger M.J. Glossmann H. Trends Pharmacol. Sci. 1998; 19: 108-115Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Both classes of drugs directly or indirectly affect Ca2+coordination to the channel's selectivity filter glutamates. It is therefore conceivable that these drugs cause Ca2+ channel block by binding to pore-forming regions, thereby altering channel gating and Ca2+ ion interaction with the pore (for review, see Refs. 2Hockerman G.H. Peterson B.Z. Johnson B.D. Catterall W.A. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 361-396Crossref PubMed Google Scholar and 3Striessnig J. Grabner M. Mitterdorfer J. Hering S. Sinnegger M.J. Glossmann H. Trends Pharmacol. Sci. 1998; 19: 108-115Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Less detailed structural information is available for the BTZ binding domain, which mediates use-dependent Ca2+channel block by diltiazem. BTZ and PAA interaction with the channel is similar in many respects; BTZs and PAAs contain a basic nitrogen essential for activity (4Kimball S.D. Hunt J.T. Barrish J.C. Das J. Floyd D.M. Lago M.W. Lee V.G. Spergel S.H. Moreland S. Hedberg S.A. Gougoutas J.Z. Malley M.F. Lau W. Bioorg. Med. Chem. 1993; 1: 285-307Crossref PubMed Scopus (16) Google Scholar) and, unlike for DHPs, Ca2+channel block critically depends on depolarization frequency ("use-dependent block"). Some early radioligand binding experiments even proposed that PAAs and BTZs compete for the same site (5Galizzi J.P. Fosset M. Lazdunski M. Biochem. Biophys. Res. Commun. 1984; 118: 239-245Crossref PubMed Scopus (44) Google Scholar) on the channel. This is supported by the finding that identical amino acid residues in IVS6 are essential for high affinity PAA as well as diltiazem sensitivity (2Hockerman G.H. Peterson B.Z. Johnson B.D. Catterall W.A. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 361-396Crossref PubMed Google Scholar, 6Hering S. Aczel S. Grabner M. Döring F. Berjukow S. Mitterdorfer J. Sinnegger M.J. Striessnig J. Degtiar V.E. Wang Z. Glossmann H. J. Biol. Chem. 1996; 271: 24471-24475Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). On the other hand several lines of evidence suggest that the PAA and BTZ binding domains are not identical; PAAs and BTZs possess a distinct structure-activity relationship for blocking (4Kimball S.D. Hunt J.T. Barrish J.C. Das J. Floyd D.M. Lago M.W. Lee V.G. Spergel S.H. Moreland S. Hedberg S.A. Gougoutas J.Z. Malley M.F. Lau W. Bioorg. Med. Chem. 1993; 1: 285-307Crossref PubMed Scopus (16) Google Scholar) and they access their binding domains from opposite sides (PAAs, the cytoplasmic side; BTZs, the extracellular side) of the channel (7Hering S. Savchenko A. Strübing C. Lakitsch M. Striessnig J. Mol. Pharmacol. 1993; 43: 820-826PubMed Google Scholar). In an extensive analysis combining information from alanine-scanning mutagenesis and radioligand binding we provide further insight into the molecular architecture of the BTZ binding domain of L-type Ca2+ channels. In particular we identified two amino acid residues in transmembrane segment IIIS6 which affect diltiazem sensitivity by different mechanisms and provide evidence for a steric interaction between the BTZ and DHP binding domain. The (+)-cis-Diastereoisomer of diltiazem was employed in all experiments (kindly provided by Göddecke (Freiburg, Germany). (+)-Isradipine was a gift from Sandoz AG (Basel, Switzerland). (+)-[3H]Isradipine (∼80 Ci/mmol) was purchased from New England Nuclear (Vienna, Austria). Benziazem was synthesized as described previously (8Kraus R. Reichl B. Kimball S.D. Grabner M. Murphy B.J. Catterall W.A. Striessnig J. J. Biol. Chem. 1996; 271: 20113-20118Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The diltiazem-sensitive α1 subunit chimera ALDIL was employed for alanine scanning mutagenesis. This chimera (Fig. 1) consists of α1A sequence with an L-type Ca2+ channel sequence transferred into the S5-S6 linkers as well as S6 segments in III and IV. Chimera ALDIL (amino acid composition: α1A1–1406, α1S965–1104 (carp skeletal muscle; GenBank accession no. M62554), α1A1544–1723, α1C1311–1437 (rabbit heart; GenBank accession no. 67515), α1A1856–2424) was constructed as follows. TheClaI-XbaI fragment of AL16 (9Grabner M. Wang Z. Hering S. Striessnig J. Glossmann H. Neuron. 1996; 16: 207-218Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) was ligated into the ClaI-XbaI linearized AL1-a (10Hering S. Aczel S. Kraus R.L. Berjukov S. Striessnig J. Timin E.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13323-13328Crossref PubMed Scopus (63) Google Scholar). Single point mutations were introduced into the SalI (nucleotide position α1S3317)-ClaI (nucleotide position α1S4925) cassette of segment IIIS6 using gene splicing by overlap extension (11Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 61-68Crossref PubMed Scopus (2648) Google Scholar). Polymerase chain reaction was performed (35 cycles, 1 min at 94 °C, 30 s at 42 °C, 1.5 min at 72 °C) using proofreading Pfu polymerase (Stratagene). Polymerase chain reaction-generated mutations were verified by sequence analysis employing the dideoxy chain termination method. Single alanine mutations were introduced into the IIIS6 segment between the following positions (amino acid positions correspond to α1C-II) (12Snutch T.P. Tomlinson W.J. Leonard J.P. Gilbert M.M. Neuron. 1991; 7: 45-57Abstract Full Text PDF PubMed Scopus (296) Google Scholar): Phe1148 to Ile1156 and Phe1158 to Phe1167. All constructs were inserted into the polyadenylating transcription plasmid pSPCBI-2. Preparation of stage V-VI oocytes from X.laevis, synthesis of capped run-off poly(A+) cRNA transcripts from XbaI-linearized cDNA templates, and injection of cRNA were described in detail previously (9Grabner M. Wang Z. Hering S. Striessnig J. Glossmann H. Neuron. 1996; 16: 207-218Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). α1 cRNAs were coinjected with β1a (13Ruth P. Roehrkasten A. Biel M. Bosse E. Regulla S. Meyer H.E. Flockerzi V. Hofmann F. Science. 1989; 245: 1115-1118Crossref PubMed Scopus (257) Google Scholar), and α2-δ subunit (14Ellis S.B. Williams M.E. Ways N.R. Brenner R. Sharp A.H. Leung A.T. Campbell K.P. McKenna E. Koch W.J. Hui A. Schwartz A. Harpold M.M. Science. 1988; 241: 1661-1664Crossref PubMed Scopus (442) Google Scholar) cRNAs. To exclude effects of endogenous Ca2+-activated Cl− currents on current kinetics, experiments were also carried out in oocytes previously injected with 50–100 nl of a 0.1 m1,2-bis(2-aminophenoxy)ethane-N, N, N, N-tetraacetic acid solution. Inward Ba2+currents (I Ba) through expressed channel complexes were measured using the two-microelectrode voltage-clamp technique (9Grabner M. Wang Z. Hering S. Striessnig J. Glossmann H. Neuron. 1996; 16: 207-218Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 15Sinnegger M.J. Wang Z. Grabner M. Hering S. Striessnig J. Glossmann H. Mitterdorfer J. J. Biol. Chem. 1997; 272: 27686-27693Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Recordings were carried out at room temperature in a bath solution containing 40 mm Ba(OH)2, 40 mm N-methyl-d-glucamine, 10 mm HEPES, 10 mm glucose, adjusted to a pH of 7.4 with methane sulfonic acid. Voltage recording and current injecting microelectrodes were filled with 2.8 m CsCl, 0.2m CsOH, 10 mm EGTA, 10 mm HEPES (adjusted to pH 7.4 with HCl) and had resistances of 0.3–2 megohm. Similar current amplitudes were obtained with mutant and ALDIL subunits. Oocytes expressing peakI Ba smaller than 400 nA or larger than 1.6 μA were excluded from analysis. Data analysis and acquisition was performed by using the pClamp software package (version 6.0, Axon Instruments). Initial "tonic" block (resting state-dependent block) was defined as peak I Ba inhibition during the first pulse after 3-min incubation at holding potential in a drug- containing solution as compared with control I Bain the absence of drug. Use (frequency)-dependent block of I Ba was measured during trains of 100-ms test pulses (0.1 Hz) applied from −80 mV to a test potential +10 mV positive to the peak of the current-voltage relationship after a 3-min equilibrium period in a drug-containing solution. To estimate the peakI Ba decay under control conditions, similar test pulses were applied in the absence of drug. Use-dependent block was expressed as the percent decrease of peakI Ba during the last pulse of the train as compared with I Ba during the first pulse. The voltage dependence of activation was determined from I-V curves obtained by step depolarizations from a holding potential of −80 mV to various test potentials. The voltage dependence of inactivation (steady state inactivation) was determined from normalized inward currents elicited during steps to +10 mV after 10-s steps to various holding potentials. The half-maximal voltage for activation (V0.5,act), and steady state inactivation (V0.5,inact) were calculated by fitting the data to the Boltzmann equation. Recovery of I Ba from inactivation was studied using a double-pulse protocol. After a 3-s depolarizing prepulse to +10 mV (holding potential −80 mV) the time course of IBarecovery was determined at −60 mV by applying 300-ms test pulses to +10 mV at various time intervals after the prepulse. PeakI Ba was normalized to the peak current amplitude measured during the prepulse. I Ba was then allowed to recover during 90 s at −100 mV. This double pulse protocol was repeated individually for each recovery time interval in the same oocyte. (+)-[3H]Isradipine binding assays were carried out at 37 °C or room temperature in 50 mmTris-HCl, pH 7.4, 0.1 mm phenylmethylsulfonyl fluoride (0.5–1 ml final assay volume). 1 μm (±)-isradipine was used to define nonspecific binding. Bound ligand was determined by filtration of the assay mixture over GF/C Whatman filters pretreated with 0.25% (v/v) polyethyleneimine for 40 min. Filters were washed three times with ice-cold buffer (20 mm Tris-HCl, pH 7.4) and then counted for radioactivity. Radioligand and protein concentrations as well as incubation conditions are given in the figure legends. To determine radioligand dissociation, (+)-[3H]isradipine was incubated (in the absence or presence of benziazem) with rabbit skeletal muscle membranes until equilibrium was reached (60 min at 22 °C). 1 μmunlabeled isradipine (in the absence or presence of unlabeled beziazem) was then added and dissociation followed for the indicated times. Dissociation rate constants were determined as the slope of the regression line from a plot of ln (fractional binding)versus time. Molecular dynamics simulations (25 °C) were performed using the SYBYL software package. Nonlinear least square fitting and statistical calculations were performed using OriginR(Microcal). Data are given as means ± S.E. for the indicated number of experiments. Student's unpaired t test was used to calculate statistical significance. We have previously shown that the photoreactive diltiazem analogue [3H]benziazem photoaffinity labels transmembrane segments IVS6 and IIIS6, suggesting that both segments are in close contact with the bound drug molecule and contain high affinity BTZ-binding determinants (8Kraus R. Reichl B. Kimball S.D. Grabner M. Murphy B.J. Catterall W.A. Striessnig J. J. Biol. Chem. 1996; 271: 20113-20118Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). We subsequently identified the binding determinants in IVS6 as Tyr1463, Ala1467, and Ile1470 (6Hering S. Aczel S. Grabner M. Döring F. Berjukow S. Mitterdorfer J. Sinnegger M.J. Striessnig J. Degtiar V.E. Wang Z. Glossmann H. J. Biol. Chem. 1996; 271: 24471-24475Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). To search for amino acid residues that affect diltiazem sensitivity in segment IIIS6 we employed alanine-scanning mutagenesis. Mutagenesis was carried out in a diltiazem and PAA-sensitive α1 subunit chimera ALDIL in which the segments corresponding to the photolabeled regions and the respective S5-S6 linkers consisted of L-type sequence, whereas the remaining sequence consisted of α1A sequence (Fig. 1, see "Experimental Procedures"). Mutated channels were coexpressed in X. laevisoocytes together with auxiliary α2-δ and β subunits and the sensitivity of the mutant channels to diltiazem was determined by using the two-microelectrode voltage-clamp technique. Mutants were screened for changes in resting-state and use-dependent block by diltiazem during a train of 15–20 depolarizing pulses (100 ms, 0.1 Hz) from a holding potential of −80 mV as described under "Experimental Procedures." As shown in Fig. 2, A and B, 100 μm diltiazem blocked about 45 ± 4.8% (n = 8) of the ALDIL-mediatedI Ba. A significant (p < 0.01) reduction in total diltiazem block was observed only after mutation of two adjacent residues, Phe1164 and Val1165, near the cytoplasmic end of IIIS6. Both residues are conserved in L- and non-L-type Ca2+ channel α1 subunits. Only 21 ± 3% (n = 12) and 8.5 ± 1.4% (n = 13) of I Ba were blocked by 100 μmdiltiazem in F1164A and V1165A, respectively. Accordingly, at lower diltiazem concentrations, block was observed for ALDIL but was almost absent for F1164A and V1165A (10 μm diltiazem; ALDIL, 30 ± 3%, n = 3; F1164A, 6 ± 2%, n = 3; V1165A, 3–5%, range,n = 2). The extent of total I Ba inhibition of the other mutants was not significantly different from ALDIL (Fig. 2 A). In mutants F1148A and I1163A, the tonic block component was increased, and the use-dependent block component significantly decreased compared with that of ALDIL. These residues may therefore comprise minor determinants for diltiazem sensitivity. We have recently shown that the sensitivity of L-type Ca2+ channels to the PAA gallopamil is decreased in slowly inactivating Ca2+channel α1 mutants (10Hering S. Aczel S. Kraus R.L. Berjukov S. Striessnig J. Timin E.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13323-13328Crossref PubMed Scopus (63) Google Scholar), suggesting that voltage-dependent inactivation is an important determinant of gallopamil sensitivity. We therefore investigated whether mutations F1164A and V1165A alter diltiazem sensitivity indirectly by changing the gating properties of the channel rather than by directly altering drug affinity for its binding domain. Fig. 3 A illustrates that V1165A (V0.5,inact = 16 ± 2 mV, n = 11), but not F1164A (V0.5,inact = 36 ± 2 mV,n = 5), caused a major (about 16 mV) shift of the V0.5,inact of the steady state inactivation curve to more positive potentials (ALDIL: V0.5,inact = 32 ± 2 mV, n = 12). At −80 mV, the holding potential selected for the experiments shown in Fig. 2, no differences in steady state inactivation were observed. Therefore it is unlikely that a higher fraction of inactivated channels at −80 mV accounted for the differences in diltiazem sensitivity in the two mutants. F1164A and V1165A also caused a significant shift of current activation (ALDIL V0.5,act = −17.3 ± 1 mV; F1164A V0.5,act = −24.7 ± 1.2 mV; V1165A V0.5,act = −10.6 ± 1.1 mV; n > 22) and slowed current inactivation during a pulse.I Ba elicited by 800-ms depolarizations from a holding potential of −80 mV to test potentials +10 mV positive to the peak of the I–V curve are shown in Fig. 3 B. Monoexponential fits of the current decay revealed an about 1.5–3-fold slower inactivation of F1164A (τ = 627 ± 118 ms, n = 10) and V1165A (τ = 1286 ± 79 ms, n = 12) than did ALDIL (τ = 447 ± 103 ms, n = 14). Taken together our data reveal that mutation of two conserved residues, Phe1164 and Val1165, on the cytoplasmic end of transmembrane segment IIIS6, change activation and inactivation gating and are major determinants for use-dependent diltiazem block. If inactivation is an important determinant for the development of use-dependent block by diltiazem, then slowing of channel inactivation in the two mutants could explain their lower sensitivity for diltiazem. To address this question the kinetics of diltiazem block and unblock were studied in more detail. The effect of 10 and 100 μmdiltiazem on the rate of current decay was determined during maintained 3-s depolarizations that allowed substantial inactivation even for the more slowly inactivating mutants F1164A and V1165A (Fig. 4 A). In ALDILcurrent decay during the 3-s test pulse was accelerated by 100 μm but not by 10 μm diltiazem (Fig. 4 A), thereby reducing I Ba at the end of the test pulse to 64 ± 9% of control (n = 4). This effect was comparable in F1164A (57 ± 6% of control,n = 5) and V1165A (68 ± 3% of control,n = 9). The drug-induced increase of the rate of I Ba decay is characteristic for drugs that block open and/or inactivated channel states during the pulse more potently than do resting channels at −80 mV. Using the extent of I Ba block by diltiazem at 3 s as a measure, the mutations did not dramatically affect channel block by 100 μm diltiazem at depolarized potentials (+10 mV). Next we investigated the effect of diltiazem on the time course of recovery of I Ba from inactivation employing a double pulse protocol. Fig. 4 B shows the recovery of I Ba for ALDIL, F1164A, and V1165A after a 3-s depolarizing conditional prepulse. Recovery was measured at −60 mV by applying 300-ms test pulses to +10 mV various periods of time after the prepulse. Peak I Ba elicited by the test pulses were normalized with respect to the peakI Ba of the prepulse (see current traces in Fig. 4 D). The recovery time courses in the absence of drug were similar for ALDIL and the two mutants (see legend to Fig. 4 B). In all cases recovery could be described by two phases.I Ba rapidly recovered monoexponentially to 80–90% of control within 15 s (termed "fast recovery"), whereas the remaining current did not recover within the 15-s period analyzed. From the total recoverable current we arbitrarily defined the contribution of "slow recovery" as the I Banot recovered after 15 s. Slow recovery accounted for 18.2 ± 0.5% (n = 5) in ALDIL, 19.4 ± 2% (n = 5) in F1164A, and 16 ± 1% (n = 9) in V1165A. This suggested that during the depolarizing prepulse all three channel constructs enter both slow ("deep") and fast inactivated states to similar extents resulting in biphasic recovery time courses. Slow inactivation has previously been reported for native and recombinant L-type Ca2+channels (16Johnson B.D. Hockerman G.H. Scheuer T. Catterall W.A. Mol. Pharmacol. 1996; 50: 1388-1400PubMed Google Scholar, 17Boyett M.R. Honjo H. Harrison S.M. Zang W.J. Kirby M.S. Pflügers Arch. 1994; 428: 39-50Crossref PubMed Scopus (42) Google Scholar). The effect of diltiazem consisted in a dramatic overall slowing of the recovery time course in ALDIL and F1164A. This was mainly due to a more than 3-fold increase of the contribution of the slowly recovering component (100 μm diltiazem: ALDIL, 70 ± 3%; F1164A, 65 ± 4%;n = 5; 10 μm diltiazem: ALDIL, 55 ± 5%; F1164A, 54 ± 2%,n = 3), whereas the time constants for the rapidly recovering components were almost unchanged (see legend to Fig. 4 B). Unlike for I Ba block development during the prepulse, the 10 μm concentrations also caused a pronounced effect on I Ba recovery of ALDIL and F1164A. As the diltiazem effect on the recovery time course was almost indistinguishable for F1164A as compared with ALDIL (Fig. 4, B and C), the smaller use-dependent block of F1164A during pulse trains (Fig. 2,A and B) is not due to a change in channel recovery but rather to the decreased inactivation during test pulses, allowing less channels to enter a (slowly recovering) drug-bound state during the pulse train (Fig. 3 B). In contrast to ALDIL and F1164A, mutation V1165A almost completely prevented this diltiazem-induced increase of the amplitude of the slowly recovering component (Fig. 4 B). The fraction of I Ba not recovered after 15 s in the presence of diltiazem (10 μm diltiazem present: 29 ± 3%, n = 5; 100 μm diltiazem present: 27 ± 3%, n = 9) was only slightly increased as compared with control (16 ± 1%, n = 9). Diltiazem delayed the time course of the fast recovering component (see legend to Fig. 4 B), which resulted in a clear inhibition of I Ba during the first few seconds. To illustrate this difference in current recovery the fraction of current recovered after 3 or 15 s in the presence of 10 μm diltiazem was divided by the fraction of current recovered in the absence of drug for ALDIL and the two mutants. Fig. 4 Cillustrates that after 3 s of recovery a comparable percentage of the current remained blocked in all constructs by 10 μmdiltiazem. This degree of block was maintained over 15 s in ALDIL and F1164A but mostly relieved in V1165A (Fig. 4 C). The acceleration of recovery from diltiazem block in mutation V1165A indicates that valine replacement either directly decreases binding affinity for the drug or allows a more rapid dissociation of the drug after being trapped in a blocked channel state. As diltiazem sensitivity in V1165A was still observed as a block of depolarized channels (Fig. 4 A) and as a block of I Ba during early recovery (Fig. 4 C), mutation V1165A does not seem to dramatically decrease diltiazem binding affinity. Earlier models describing block of L-type Ca2+ channels by PAA Ca2+ antagonists suggest that use-dependent Ca2+ channel blockers bind to open channels, but are trapped within inactivated channels and require removal of inactivation for rapid dissociation and unblocking (10Hering S. Aczel S. Kraus R.L. Berjukov S. Striessnig J. Timin E.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13323-13328Crossref PubMed Scopus (63) Google Scholar, 16Johnson B.D. Hockerman G.H. Scheuer T. Catterall W.A. Mol. Pharmacol. 1996; 50: 1388-1400PubMed Google Scholar). We therefore propose that replacement of the bulkier valine in position 1165 by an alanine (partially) removes a dissociation barrier and facilitates dissociation of diltiazem from blocked (presumably inactivated) channel states. If this were true then we would expect that bulky diltiazem derivatives would escape at a slower rate. As a consequence such a bulky drug should, at least partially, overcome the effect of the mutation and cause substantial use-dependent block even in V1165A. We tested this hypothesis using the bulkier diltiazem derivative benziazem, which has previously been shown to photoaffinity label segment IIIS6 (8Kraus R. Reichl B. Kimball S.D. Grabner M. Murphy B.J. Catterall W.A. Striessnig J. J. Biol. Chem. 1996; 271: 20113-20118Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Benziazem contains a bulky benzophenone substituent remote from the pharmacophores important for interaction with the benzothiazepine binding domain (8Kraus R. Reichl B. Kimball S.D. Grabner M. Murphy B.J. Catterall W.A. Striessnig J. J. Biol. Chem. 1996; 271: 20113-20118Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Fig. 5 shows that the extent of block by 10 and 100 μm benziazem (gray columns) was only slightly (1.3–1.5-fold) larger than by diltiazem in ALDIL. In contrast, under the same experimental conditions benziazem caused 3.6–4.5-fold larger block of V1165A at both concentrations than did diltiazem, indicating that this bulkier drug must still be able to considerably slow recovery from inactivation in V1165A. The above finding also prompted us to investigate whether the larger size of benziazem can alter its noncompetitive interaction mechanism with the DHP binding domain, which is also formed predominantly by IIIS6 residues (2Hockerman G.H. Peterson B.Z. Johnson B.D. Catterall W.A. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 361-396Crossref PubMed Google Scholar, 3Striessnig J. Grabner M. Mitterdorfer J. Hering S. Sinnegger M.J. Glossmann H. Trends Pharmacol. Sci. 1998; 19: 108-115Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). We compared the effects of the bulky benziazem and its analogues Bz-BAZ and DMBODIPY-BAZ (18Brauns T. Cai Z.-W. Kimball D. Kang H.-C. Haugland R.P. Berger W. Berjukov S. Hering S. Glossmann H. Striessnig J. Biochemistry. 1995; 34: 3461-3469Crossref PubMed Scopus (9) Google Scholar) with the smaller molecules diltiazem and SQ32,910 (7Hering S. Savchenko A. Strübing C. Lakitsch M. Striessnig J. Mol. Pharmacol. 1993; 43: 820-826PubMed Google Scholar) on (+)-[3H]isradipine binding to skeletal muscle L-type Ca2+ channels. Fig. 6 A illustrates that, in contrast to (+)-cis-diltiazem and SQ32,910, which are known stimulators of equilibrium (+)-[3H]isradipine binding (7Hering S. Savchenko A. Strübing C. Lakitsch M. Striessnig J. Mol. Pharmacol. 1993; 43: 820-826PubMed Google Scholar), all three bulky derivatives were inhibitory. For benziazem and Bz-BAZ incomplete inhibition could be demonstrated (maximal inhibition to 73 ± 3% (n = 10) and 89 ± 2% (n = 3) of control). Benziazem was selected to further investigate the inhibitory mechanism of these compounds. Saturation analysis (Fig. 6 B) revealed that 1 μmbenziazem decreased the apparent binding affinity of the DHP by about 2.8-fold (control, K d = 0.43 ± 0.06 nm; 1 μm benziazem present,K d = 1.2 ± 0.06 nm;n = 3) without major change in B max (control, 179 ± 22 pm; 1 μm benziazem present, 154 ± 13 pm;n = 3). This affinity decrease was mainly due to a destabilization of the DHP·Ca2+ channel complex as revealed by dissociation kinetics. For dissociation experiments (Fig. 6 C) (+)-[3H]isradipine was incubated in the absence (control) and presence of 1 μm benziazem until binding equilibrium was reached. (+)-[3H]Isradipine dissociation from control and ternary channel complexes was then induced by an excess of unlabeled isradipine ("cold chase"). As shown in Fig. 6 C dissociation was about 2-fold faster from ternary complexes than from channels preincubated without benziazem. This indicated that the increase of k −1mainly accounts for the increase in K d(K d =k −1/k +1) (k −1 = dissociation rate constant;k +1 = association rate constant). However, this destabilizing effect of benziazem was not seen when 1 μm(or 10 μm, not shown) benziazem was added (together with unlabeled (+)-isradipine, "double chase") after the (+)-[3H]isradipine channel complex had already been formed (Fig. 6 D). These experiments clearly show that benziazem destabilized DHP binding in the ternary complex but that the DHP must dissociate before this effect can occur. In control experiments (+)-verapamil, which is known to inhibit DHP binding via an allosteric mechanism (1Glossmann H. Striessnig J. Rev. Physiol. Biochem. Pharmacol. 1990; 114: 1-105Crossref PubMed Google Scholar), also partially inhibited (+)-[3H]isradipine equilibrium binding but, in contrast to benziazem, was also able to accelerate (+)-[3H]isradipine dissociation from preformed high affinity (+)-[3H]isradipine-channel complexes (Fig. 6 D). The benziazem binding data are difficult to interpret on the basis of an allosteric model, which would predict a destabilizing effect of an unlabeled drug through binding to a separate, allosterically coupled site in both types of dissociation experiments (as observed for (+)-verapamil). Modulation of (+)-[3H]isradipine binding by benziazem is best explained as a steric interaction between the bound benziazem and DHP molecule, in which the bulky side chain of benziazem can decrease DHP binding stability once the DHP is dissociated. Although a precise mechanism for this effect cannot be derived from our experiments it is likely that this is caused by a protrusion of the bulky benziazem side chain into the DHP binding region. On the basis of this proposed steric mechanism we can use benziazem and its bulky analogues as molecular rulers. From the distance of their side chains from the basic amine pharmacophore as calculated from molecular dynamics simulations (benziazem ≤ 17 Å; DMBODIPY-BAZ ≤ 17 Å; Bz-BAZ ≤ 11 Å) we can estimate the maximal distance between the BTZ binding domain and the bound DHP molecule to be 11–17 Å. Our study provides insight into the molecular mechanism of diltiazem interaction with L-type Ca2+ channels and the molecular organization of the Ca2+ antagonist binding domains on α1 subunits. Using alanine scanning mutagenesis we identified amino acid residues Phe1164 and Val1165 at the cytoplasmic end of the putative IIIS6 α-helix as important determinants for diltiazem sensitivity. Although our functional studies could not demonstrate their direct contribution to the formation of the diltiazem binding pocket, these residues indirectly control diltiazem sensitivity by slowing channel inactivation (both residues, Fig. 3 B) and by facilitating channel recovery from drug block (V1165A, Fig. 4). We also provide evidence that introduction of bulky side chains into the diltiazem molecule (i) largely prevented the V1165A mutational effect by stabilizing channel block and (ii) produced compounds noncompetitively inhibiting rather than stimulating DHP binding to the channel through an apparently steric interaction mechanism. Among the 19 mutations investigated, mutants F1164A and V1165A caused the most pronounced effects on channel inactivation kinetics as well as diltiazem sensitivity. Our studies do not exclude the possibility that minor effects on diltiazem sensitivity are present in the other mutants which will, however, require a more detailed analysis. Phe1164 and Val1165 are highly conserved in all high voltage-activated Ca2+ channel α1subunits. As mutation of these residues not only affects inactivation gating in ALDIL but also in α1C (19Hockerman G.H. Johnson B.D. Abbott M.R. Scheuer T. Catterall W.A. J. Biol. Chem. 1997; 272: 18759-18765Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) it is likely that they are part of a common inactivation mechanism in all Ca2+ channel α1 subunits. Our experiments provide important insight into the mechanism of diltiazem block. It promotes a slowly recovering channel state that explains the development of use-dependent block at a higher depolarization frequency. This effect of diltiazem is qualitatively indistinguishable from the mechanism of action of phenylalkylamines (10Hering S. Aczel S. Kraus R.L. Berjukov S. Striessnig J. Timin E.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13323-13328Crossref PubMed Scopus (63) Google Scholar, 16Johnson B.D. Hockerman G.H. Scheuer T. Catterall W.A. Mol. Pharmacol. 1996; 50: 1388-1400PubMed Google Scholar). Although different kinetic models have been proposed to account for this effect (10Hering S. Aczel S. Kraus R.L. Berjukov S. Striessnig J. Timin E.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13323-13328Crossref PubMed Scopus (63) Google Scholar, 16Johnson B.D. Hockerman G.H. Scheuer T. Catterall W.A. Mol. Pharmacol. 1996; 50: 1388-1400PubMed Google Scholar), it is believed that the time course of recovery from channel block is critically determined by steric factors governing the rate of dissociation of the drug trapped within inactivated channel states (10Hering S. Aczel S. Kraus R.L. Berjukov S. Striessnig J. Timin E.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13323-13328Crossref PubMed Scopus (63) Google Scholar, 16Johnson B.D. Hockerman G.H. Scheuer T. Catterall W.A. Mol. Pharmacol. 1996; 50: 1388-1400PubMed Google Scholar, 20Hille B. Ionic Channels of Excitable Membranes. Sinauer, Sunderland, MA1991Google Scholar). The intriguing effect of mutation V1165A was to allow the channel to recover from diltiazem block more rapidly. A possible molecular explanation for this finding is that Val1165 as part of an inactivation mechanism also controls diltiazem dissociation from inactivated channels. Replacement by a less bulky alanine would therefore not only affect inactivation but also facilitate drug dissociation thereby accelerating recovery from drug block. Such a steric mechanism also predicts that not only the size of the "dissociation pathway" generated by the mutation but also the size of the drug itself affects the rate of dissociation. The fact that the bulkier diltiazem derivative benziazem still was an effective use-dependent blocker of V1165A (Fig. 5) strongly supports the above model. Together with its slower inactivation time course the accelerated recovery from diltiazem block of V1165A also explains the dramatic decrease of its use-dependent block at higher depolarization frequency (Fig. 2). We describe another interesting difference between diltiazem and its bulkier derivatives benziazem, DMBODIPY-BAZ, and BZ-BAZ. They act as partial inhibitors rather than stimulators of (+)-[3H]isradipine binding to L-type Ca2+channels. A detailed kinetic analysis of the benziazem effect led us to conclude that partial DHP binding inhibition by the bulky side chains is based on a steric rather than allosteric decrease of (+)-[3H]isradipine binding affinity. Our results are in accordance with our previous detailed spectroscopic analysis of the interaction of fluorescently labeled diltiazem derivative, DMBODIPY-BAZ, with unlabeled DHPs (21Brauns T. Prinz H. Kimball S.D. Haugland R.P. Striessnig J. Glossmann H. Biochemistry. 1997; 36: 3625-3631Crossref PubMed Scopus (16) Google Scholar). We found evidence for a direct drug-drug interaction between channel bound DHPs and DMBODIPY-BAZ, also supporting a steric model. From a quantitative analysis of high time resolution kinetic binding data (21Brauns T. Prinz H. Kimball S.D. Haugland R.P. Striessnig J. Glossmann H. Biochemistry. 1997; 36: 3625-3631Crossref PubMed Scopus (16) Google Scholar) we predicted a 3-fold decrease of (+)-isradipine affinity after DMBODIPY-BAZ binding. This is now confirmed directly employing (+)-[3H]isradipine as a radioligand supporting a model in which the DHP and BTZ binding domain are located in close proximity between IIIS6 and IVS6 in the folded α1 subunit structure. Using the bulky side chains of diltiazem analogues as molecular rulers we currently estimate a maximal distance of 11–17 Å between the basic nitrogen of bound BTZs and the bound DHP molecule. This is about 2 times the narrowest diameter of the channel pore (≈6 Å) (22McCleskey E.W. Almers W. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7149-7153Crossref PubMed Scopus (183) Google Scholar). It will be interesting to investigate the behavior of compounds with even smaller side chains (23Kimball S.D. Floyd D.M. Das J. Hunt J.T. Krapcho J. Rovnyak G. Duff K.J. Lee V.G. Moquin R.V. Turk C.F. Hedberg S.A. Moreland S. Brittain R.J. McMullen D.M. Normandin D.E. Cucinotta G.G. J. Med. Chem. 1992; 35: 780-793Crossref PubMed Scopus (45) Google Scholar) on DHP binding, which may help to get a more precise estimate of the minimal distance between the two bound drug molecules. Our and previous data reveal several important common properties between PAA and BTZs with respect to channel interaction and modulation. First, they both share residues Tyr1463, Ala1467, and Ile1470 in segment IVS6 as common binding motifs (6Hering S. Aczel S. Grabner M. Döring F. Berjukow S. Mitterdorfer J. Sinnegger M.J. Striessnig J. Degtiar V.E. Wang Z. Glossmann H. J. Biol. Chem. 1996; 271: 24471-24475Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 24Hockerman G.H. Johnson B.D. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 22119-22122Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar); second, Val1165 in IIIS6 affects not only PAA (19Hockerman G.H. Johnson B.D. Abbott M.R. Scheuer T. Catterall W.A. J. Biol. Chem. 1997; 272: 18759-18765Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) but also BTZ sensitivity (this study); third, despite a clearly distinct structure-activity relationship of PAAs and BTZs both classes of drugs exhibit a similar mechanism of use-dependent block of L-type Ca2+ channels, namely by inducing slow recovery from inactivation. Rather than distinguishing between two separate binding domains for PAAs and BTZs as proposed by an allosteric model (1Glossmann H. Striessnig J. Rev. Physiol. Biochem. Pharmacol. 1990; 114: 1-105Crossref PubMed Google Scholar) a common or strongly overlapping binding domain mostly formed by IIIS6 and IVS6 residues should be proposed through which cationic amphiphilic PAAs and BTZs modulate L-type channel activity. We thank Dr. S. Berjukov for helpful discussion, D. Kandler and B. Kurka for expert technical assistance, Drs. Y. Mori and K. Imoto for α1A cDNA, and Dr. T. Langer for molecular dynamics simulations. We thank H. Glossmann for continuous support.
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