Sequence Differences between alpha 1C and alpha 1S Ca2+ Channel Subunits Reveal Structural Determinants of a Guarded and Modulated Benzothiazepine Receptor*

Stanislav BerjukowDagger , Franz GappDagger , Stefan Aczél, Martina J. Sinnegger, Joerg Mitterdorfer, Hartmut Glossmann, and Steffen Hering§

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

    ABSTRACT
Top
Abstract
Introduction
References

The molecular basis of the Ca2+ channel block by (+)-cis-diltiazem was studied in class A/L-type chimeras and mutant alpha 1C-a Ca2+ channels. Chimeras consisted of either rabbit heart (alpha 1C-a) or carp skeletal muscle (alpha 1S) sequence in transmembrane segments IIIS6, IVS6, and adjacent S5-S6 linkers. Only chimeras containing sequences from alpha 1C-a were efficiently blocked by (+)-cis-diltiazem, whereas the phenylalkylamine (-)-gallopamil efficiently blocked both constructs. Carp skeletal muscle and rabbit heart Ca2+ channel alpha 1 subunits differ with respect to two nonconserved amino acids in segments IVS6. Transfer of a single leucine (Leu1383, located at the extracellular mouth of the pore) from IVS6 alpha 1C-a to IVS6 of alpha 1S significantly increased the (+)-cis-diltiazem sensitivity of the corresponding mutant L1383I. An analysis of the role of the two heterologous amino acids in a L-type alpha 1 subunit revealed that corresponding amino acids in position 1487 (outer channel mouth) determine recovery of resting Ca2+ channels from block by (+)-cis-diltiazem. The second heterologous amino acid in position 1504 of segment IVS6 (inner channel mouth) was identified as crucial inactivation determinant of L-type Ca2+ channels. This residue simultaneously modulates drug binding during membrane depolarization. Our study provides the first evidence for a guarded and modulated benzothiazepine receptor on L-type channels.

    INTRODUCTION
Top
Abstract
Introduction
References

Voltage-gated Ca2+ channels mediate Ca2+ currents in muscle, endocrine cells, and neurons. They regulate a variety of important cell functions such as contractility, excitability, secretion, and gene expression. A distinct feature of L-type Ca2+ channels is their high affinity for Ca2+ antagonists (1). The stereoselective, high affinity drug receptors for Ca2+ antagonists such as 1,4-dihydropyridines, phenylalkylamines (PAAs),1 and benzothiazepines (BTZs) are located on the pore-forming alpha 1 subunit of L-type channels (classes C, D, and S) (for review see Refs. 2 and 3).

Recent studies on cloned Ca2+ channel alpha 1 subunits provided first insights into the molecular architecture of the drug binding domains for Ca2+ antagonists. In a "loss of function" approach receptor determinants of Ca2+ antagonists were identified by replacing putative drug binding sequences by corresponding sequence stretches of non-L-type channels or by systematic alanine scanning mutagenesis (4-7). Alternatively, in a "gain of function" approach, 1,4-dihydropyridine, PAA, and BTZ sensitivity could be transferred to alpha 1A or alpha 1E subunits by exchanging portions of non-L-type sequences or even single amino acid residues with the corresponding L-type counterparts (8-12). The crucial structural elements of the Ca2+ antagonist-binding domains involve amino acids in transmembrane segments IIIS5, IIIS6, and IVS6. Adjacent IIIS5-IIIS6 and IVS5-IVS6 linkers contain additional, less crucial drug binding determinants (see Ref. 3 for review).

We have previously demonstrated that transfer of the three high affinity determinants of the PAA receptor (Tyr1804, Ala1808, and Ile1811; see Ref. 11) to segment IVS6 of the alpha 1A subunit enhances the BTZ sensitivity of the resulting class A channel mutant (Ref. 12; see also Ref. 13). Recent photoaffinity labeling experiments by Kraus et al. (14) suggest additional BTZ receptor determinants in the segment IIIS6 of L-type channels (see also Ref. 15).

Here, we compare the PAA and BTZ sensitivity of two class A/L-type Ca2+ channel chimeras consisting of L-type sequence in transmembrane segments IIIS6 and IVS6 and adjacent S5-S6 linkers that was derived from either carp skeletal muscle alpha 1S (chimera AL1) or the rabbit heart alpha 1C-a (AL16) and introduced into the sequence background of alpha 1A (Fig. 1A). From previous work (12-14, 16) chimeras AL1 (8) and AL16 (12, 17) were expected to carry the determinants for high affinity BTZ binding in their L-type segments IIIS6 and IVS6. Surprisingly, only the chimeric channel containing sequence stretches of the cardiac alpha 1C-a subunit was efficiently blocked by (+)-cis-diltiazem. Sequence comparison of alpha 1S and alpha 1C-a revealed two nonconserved amino acids in segment IVS6. Transfer of a single amino acid (Ile1487) from segment IVS6 of the cardiac alpha 1C-a sequence to the carp skeletal muscle alpha 1S enhanced BTZ sensitivity of the resulting mutant to the level of the alpha 1A/alpha 1C-a chimera. The second divergent amino acid (Val1504, alpha 1C-a numbering) was identified as a strong class C channel inactivation determinant.

BTZ blocks Ca2+ channels in a use-dependent manner (18). The structural basis of state-dependent Ca2+ channel block by BTZ is not sufficiently understood. Use-dependent block can be attributed to state-dependent removal of guarding structures or high affinity drug binding to open or inactivated channels during membrane depolarization (19, 20).

A detailed analysis of the impact of both amino acids for Ca2+ channel block of alpha 1C-a mutants enabled not only the identification of additional determinants of the diltiazem receptor site but also provided new insights into the molecular mechanism of L-type Ca2+ channel block by BTZ. Our data suggest that besides the putatively pore orientated amino acids in the central part of segment IVS6 (12), the BTZ receptor contains an additional receptor determinant that is located near the extracellular channel mouth. Furthermore, a residue in segment IVS6 (Val1504) that is localized close to the intracellular mouth of the pore and determines inactivation also appears to modulate drug binding. We obtained first structural evidence for a guarded and modulated BTZ receptor of L-type channels.

    EXPERIMENTAL PROCEDURES

Chimeric Class A/L-type Channel Constructs-- Chimeras AL1 and AL16 were generated by replacing the transmembrane segments IIIS6 and IVS6 and the adjacent S5-S6 linkers of the rabbit brain class A Ca2+ channel (BI-2) alpha 1A (21) by corresponding sequences of either the carp skeletal muscle alpha 1S (AL1; see Ref. 8) or the rabbit cardiac alpha 1C-a (AL16; see Ref. 17).

Mutant alpha 1 Subunits-- Mutant L1383I (amino acid numbering according to alpha 1S; see Ref. 22) was constructed by introducing point mutations into alpha 1S-cDNA (8). cDNAs were inserted into a KpnI*-BglII-cassette of chimera AL1 (nucleotides 5467 and 6185, numbering according to alpha 1A). The KpnI* site was generated by introducing a silent mutation. I1487L, I1487A, and V1504A were introduced into alpha 1C-a-cDNA of the L-type construct Lc using a EcoRV-BstEII-cassette (nucleotides 4542 and 4833). Lc is a construct corresponding to alpha 1C-a-cDNA (23) with part of the amino terminus replaced by carp alpha 1S sequence (to increase expression density; see Ref. 8) containing two additional restriction sites (HindIII/2955; SalI/3670). Amino acid and nucleotide numbering is according to alpha 1C-a (see Ref. 23).

All mutations were introduced by using polymerase chain reaction, which was performed with proof-reading Pfu polymerase (Stratagene). Fragments amplified by polymerase chain reaction were sequenced entirely to confirm sequence integrity.

Electrophysiology-- Two microelectrode voltage-clamp of Xenopus oocytes was performed 2-7 days after microinjection of cRNAs in approximately equimolar mixtures of alpha 1 (0.3 ng/50 nl)/beta 1a (0.1 ng/50 nl)/alpha 2delta (0.2 ng/50 nl) as described previously (8). All experiments were carried out at room temperature in bath solution with the following composition: 40 mM Ba(OH)2, 40 mM N-methyl-D-glucamine, 10 mM HEPES, 10 mM glucose (pH adjusted to 7.4 with methanesulfonic acid). 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 MOmega . Activation of endogenous Ca2+-activated Cl- conductance by barium influx through Ca2+ channels was eliminated by injecting 50-100 nl of a 0.1 M 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid solution (adjusted with methanesulfonic acid to pH 7.0) into oocytes 20-40 min before the experiments. The recording chamber (150 µl of total volume) was continuously perfused at a flow rate of 1 ml/min with control or drug-containing solutions. The pClamp software package (version 6.0, Axon Instruments, Inc.) was used for data acquisition and analysis. Data were digitized at 2 kHz, filtered at 1 kHz, and stored on a computer hard disk. Leakage current correction was performed by using average values of scaled leakage currents elicited by a 10 mV hyperpolarizing voltage step.

Tonic ("resting state-dependent") block was measured as peak IBa inhibition during the first pulse after a 3-min equilibration in drug-containing solution at rest (holding potential -80 mV), and use-dependent current inhibition was subsequently estimated during 0.1 or 1 Hz trains of 100-ms test pulses. Inactivation of IBa during a pulse train was estimated by applying similar test pulses in the absence of drug.

Recovery of IBa from inactivation was studied at -80 mV by depolarizing Ca2+channels during a 3-s prepulse to 20 mV and subsequent application of a second test pulse to 20 mV at various time intervals after the conditioning prepulse. Peak IBa values were normalized to the peak current measured during the prepulse. IBa recovered between 90 and 100% during a subsequent 3-min rest at -100 mV. The time course of IBa recovery from inactivation was fit to single or biexponential functions (IBa, recovery = A*exp(-t/tau rec, fast) B*exp(-t/tau rec, slow) + C). Data are given as the means ± S.E. Statistical significance of IBa block by diltiazem or gallopamil compared with current decay under control conditions was calculated according to unpaired Student t test (p < 0.05).

    RESULTS

Newly Identified Determinants of Ca2+ Channel Block by (+)-cis-Diltiazem in Segment IVS6-- Fig. 1A shows a schematic representation of the class A/L-type chimeras AL1 and AL16 consisting of either L-type sequence of carp skeletal muscle alpha 1S (AL1) or rabbit heart alpha 1C-a (AL16) in segments IIIS6 and IVS6 and adjacent S5-S6 loops. Previously identified determinants of the BTZ receptor site in the central part of the putative pore-lining alpha -helical IVS6 segment (Tyr1386, Ala1390, and Ile1393; see Refs. 12 and 13) are highlighted in Fig. 1B. The amino acid sequences of alpha 1C-a and alpha 1S IVS6 segments are identical with the exception of two nonconserved amino acid residues (Fig. 1C). Leu1383 right-arrow Ile and Ile1400 right-arrow Val represent conservative substitutions as each of the residues is nonpolar, hydrophobic, and of comparable size. As shown in Fig. 1D, corresponding IBa values of chimeras AL1 and AL16 displayed different kinetic properties with chimera AL1 inactivating significantly faster (52 ± 1% IBa inactivation during a 300-ms pulse from -80 to 20 mV, n = 18) than AL16 (31 ± 2%, n = 13, p < 0.001) (Fig. 1D).


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Fig. 1.   Schematic representation of class A/L-type Ca2+ channel chimeras AL1 and AL16. A, class A (alpha 1A) sequence is shown as white transmembrane segments and L-type sequences from carp skeletal muscle (alpha 1S, AL1) or rabbit heart (alpha 1C-a, AL16) in segments IIIS6 and IVS6, and adjacent S5-S6 connecting loops are illustrated as black segments and bold lines. B, schematic alpha -helical representation of amino acid sequence of segment IVS6 (alpha 1S) of chimera AL1 (light gray). High affinity PAA/BTZ determinants (Tyr1386, Ala1390, and Ile1393; Refs. 11 and 12), and heterologous residues of skeletal muscle alpha 1S and rabbit heart alpha 1C-a sequences in positions 1383 and 1400 are highlighted in black. C, sequence alignment of transmembrane segments IVS6 of chimeras AL1 (carp skeletal muscle alpha 1S, see Ref. 32) and AL16 (cardiac L-type alpha 1C-a; see Ref. 17). High affinity determinants of the PAA receptor site (open boxes) and sequence differences between alpha 1C-a and alpha 1S (black boxes) are highlighted. D, IBa of AL1 and AL16 during depolarizing test pulses from -80 mV to the indicated test potentials.

Our recent finding that inactivation determinants in pore lining S6 segments affect use-dependent Ca2+ channel block by PAA (24) prompted us to examine possible differences in PAA and BTZ sensitivity between chimeras AL1 and AL16 (Fig. 1D). Drug sensitivity was characterized as resting state and use-dependent IBa inhibition (see also Refs. 12 and 25).

Despite the presence of L-type sequence in both constructs (Fig. 1A), (+)-cis-diltiazem (100 µM) induced much less IBa inhibition of AL1 compared with the pronounced block of AL16 (Fig. 2, A and B). Fig. 2 compares IBa block of AL1 and AL16 by (-)-gallopamil and (+)-cis-diltiazem. In line with previous observations, (-)-gallopamil (100 µM) induced pronounced use-dependent Ca2+ channel block of AL1 and AL16 (12, 16). Resting state-dependent Ca2+ channel block was more prominent for the benzothiazepine than for the PAA (Fig. 2B; see Ref. 18 for similar observations on cardiac myocytes).


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Fig. 2.   Sensitivity of AL1 and AL16 for (+)-cis-diltiazem and (-)-gallopamil. A, use-dependent block of IBa in AL1 (upper panel) and AL16 (lower panel) by (+)-cis-diltiazem and (-)-gallopamil was estimated during trains of 15 test pulses (100 ms) applied from -80 mV to 20 mV at a frequency of 0.1 Hz. Identical pulse protocols were applied in the absence of drug (control). B, use-dependent IBa inhibition (in percentages, means ± S.E., n = 4-9) during 15 pulses (see A) by 100 µM (+)-cis-diltiazem (shaded columns) or 100 µM (-)-gallopamil (hatched columns) compared with current decay in the absence of drug (white columns) at a holding potential of -80 mV. Resting state-dependent block (IBa inhibition during the first pulse in drug; black columns) was more pronounced for AL16. C and D, IBa recovery from inactivation of AL1 (C) and AL16 (D) were measured by a two-pulse protocol in the absence (open symbols) and presence (filled symbols) of 100 µM (+)-cis-diltiazem. The currents were inactivated by 3-s prepulses to 20 mV. IBa recovery at -80 mV was measured by applying a sequence of test pulses at various times after the prepulse (see "Experimental Procedures"). After a two-pulse experiment the membrane was hyperpolarized to -100 mV for 4 min to permit recovery from block and inactivation. Continuous lines are biexponential fits to the mean time courses, with AL1 recovery time constants tau rec, fast (open circle ) = 1.9 ± 0.3 (n = 4) and tau rec, slow (open circle )= 31 ± 6 (n = 4) versus tau rec, fast () =1.1 ± 0.2 (n = 4) and tau rec, slow () = 35 ± 3 (n = 4) (C) and AL16 recovery time constants tau rec, fast (open circle ) = 2.1 ± 0.3 (n = 4) and tau rec, slow (open circle ) = 25 ± 4 (n = 4) versus tau rec, fast () = 1.2 ± 0.2 (n = 4) and tau rec, slow () = 63 ± 10 (n = 4) (D).

Thus, the results shown in Fig. 2A suggest that crucial determinants of the BTZ receptor are missing in the alpha 1S sequence of AL1. Differences in (+)-cis-diltiazem sensitivity were further analyzed by comparing the time course of IBa recovery at rest. Diltiazem slowed IBa recovery mainly by delaying the slow recovery component (tau rec, slow) (Fig. 2, C and D). In line with the more pronounced use-dependent block, IBa of AL16 recovered at a significantly slower rate from block by the benzothiazepine than IBa of AL1 (tau rec, slow (AL1) = 35 ± 3 s versus tau rec, slow (AL16) = 63 ± 10 s, Fig. 2, A, C, and D). The fast recovery time constant was not significantly changed (tau rec, fast (AL1) = 1.1 ± 0.2 s versus tau rec, fast (AL16) = 1.2 ± 0.2 s).

Transfer of a BTZ Receptor Determinant from alpha 1C-a to IVS6 of alpha 1S-- To elucidate the structural basis for the higher BTZ sensitivity of AL16 (Fig. 2), we transferred Ile1383 from alpha 1C-a to the alpha 1S-derived IVS6 segment of AL1 and analyzed the sensitivity of the resulting mutant (L1383I) for (+)-cis-diltiazem.

As shown in Fig. 3, mutation L1383I (Fig. 1B) significantly enhanced the BTZ sensitivity of AL1. The single leucine to isoleucine substitution in segment IVS6 of AL1 induced the same amount of resting state and use-dependent block by (+)-cis-diltiazem (100 µM) as in the highly sensitive chimera AL16 (Fig. 3A). The IC50 value of use-dependent IBa inhibition of AL16 (IC50 = 70 ± 15 µM) did not significantly differ from the mutant L1381I (IC50 = 83 ± 20 µM, p > 0.05) but was significantly different from AL1 (IC50 = 880 ± 65 µM, n >=  3 for each concentration).


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Fig. 3.   Transfer of BTZ sensitivity from IVS6 of alpha 1C-a (AL16) to IVS6 of alpha 1S (AL1). A, use-dependent IBa inhibition by (+)-cis-diltiazem (100 µM) of AL1, L1383I and AL16. Channel block was estimated as described in the legend to Fig. 2A. B, IBa recovery from inactivation of L1383I was estimated as shown in panels C and D of Fig. 2. open circle , control; , drug. Continuous lines are biexponential fits to the mean time courses, with L1383I recovery time constants tau rec, fast (open circle ) = 2.6 ± 0.4 s and tau rec, slow (open circle ) = 39 ± 7 s (n = 4) versus tau rec, fast () = 1.2 ± 0.3 s and tau rec, slow () = 59 ± 7 s (n = 4).

Furthermore, recovery of L1383I in the presence of (+)-cis-diltiazem was slower than in AL1 and comparable with recovery of AL16 (tau rec, slow (L13831I) = 59 ± 7 s, Figs. 2C and 3B). Mutation L1383I transferred not only the sensitivity for (+)-cis-diltiazem but additionally slowed inactivation kinetics compared with AL1 (see inset in Fig. 3A, 27 ± 3% during 300-ms test pulse to 20 mV, n = 6, significantly slower than AL1). The later finding was also evident from a reduced accumulation of Ca2+ channels in inactivation during control pulse trains (white columns in Fig. 3A).

Taken together, the rearrangement of a single methyl group in the outer part of segment IVS6 (mutation L1383I) increased sensitivity for (+)-cis-diltiazem by delaying unblock of resting Ca2+ channels. A crucial role of Ile1383 for block of resting Ca2+ channels by (+)-cis-diltiazem is further supported by an enhanced resting state-dependent block of L1383I compared with AL1 (Fig. 3A).

Role of Ile1487 and Val1504 in (+)-cis-Diltiazem Block of a Cardiac L-type alpha 1 Subunit-- Results in Fig. 3 illustrate that an amino acid at the outer channel mouth in segment IVS6 may have a specific effect on (+)-cis-diltiazem binding and dissociation to resting (closed) channels. Because this finding was obtained using chimeric channels, which might produce peculiar effects caused by an altered sequence environment, we subsequently studied the role of both divergent amino acids in segment IVS6 (Ile1383 and Val1400) using the cardiac alpha 1C-a subunit derived construct Lc (see "Experimental Procedures"). As shown in Fig. 4, substitution of the "cardiac" isoleucine in position 1487 by either the corresponding leucine of the carp skeletal muscle alpha 1S or an alanine (I1487L or I1487A) significantly reduced use-dependent channel block by accelerating unblock at rest (Fig. 4, compare unblock of I1487A with unblock of Lc).


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Fig. 4.   Use-dependent block and recovery of Lc and mutant Lc subunits I1487L, I1487A, and V1504A. A, comparison of the inactivation time course of Lc and mutants I1487L, I1487A, and V1504A illustrated during 1-s depolarizing pulses from -80mV to the indicated test potentials. Mutation V1504A substantially reduced channel inactivation. Compare with inactivation kinetics of Lc. B, comparison of use-dependent IBa block of Lc and derived mutants. IBa inhibition was measured as cumulative current inhibition (in percentages) during 15 depolarizing pulses (100 ms) after 3-min incubation of the Xenopus oocytes in drug (shaded columns) (see Fig. 2B). Test pulses were applied at a frequency of 1 Hz. Resting state-dependent block shown by the black columns. Peak IBa decay under control conditions is shown by the white columns. Bars represent the mean ± S.E. (n = 4-8). Statistical significance compared with IBa block of Lc is indicated by asterisks. C, IBa recovery from inactivation in the absence or presence of 100 µM (+)-cis-diltiazem. Continuous lines are single exponential fits to the mean recovery time courses of Lc (open circle , control; , in drug), I1487A (black-triangle, in drug), and V1504A (black-diamond , in drug) with recovery time constants: Lc (control) tau rec = 0.18 ± 0.04 s (n = 4) versus Lc (drug) tau rec = 2.94 ± 0.32 s (n = 4), I1487L (control, not shown) tau rec = 0.31 ± 0.12 s (n = 4) versus I1487A (drug) tau rec = 1.0 ± 0.06 s (n = 4), I1487A (control, not shown) tau rec = 0.27 ± 0.12 s (n = 5) versus I1487A (drug) tau rec = 0.97 ± 0.13 s (n = 6), I1504A (control, not shown) tau rec = 9.93 ± 1.68 s (n = 3) versus I1504A (drug) tau rec = 3.5 ± 0.55 s (n = 3). D, mean time constants of IBa recovery from inactivation in control (white columns) compared with recovery time constants in the presence of 100 µM (+)-cis-diltiazem (black columns) of constructs Lc, I1487L, I1487A, and V1504A. Because of its slower inactivation (see panel A), recovery of mutant V1504A was estimated after 30 s prepulses. Statistical significance compared with IBa block of Lc is indicated by asterisks.

Substituting alanine for valine in position 1504 at the inner channel mouth (V1504A) dramatically slowed Ca2+ channel inactivation (Fig. 4A) and simultaneously diminished use-dependent IBa inhibition by (+)-cis-diltiazem (Fig. 4B). A subsequent analysis of the IBa recovery kinetics from block clearly demonstrated that this substitution at the inner channel mouth (V1504A) did not significantly affect unblock of resting channels (Fig. 4C, compare unblock of V1504A and Lc).

Kinetics of Open Ca2+ Channel Block by (+)-cis-Diltiazem-- To investigate the kinetics of (+)-cis-diltiazem interaction with Ca2+ channels in the open state, we analyzed the rate and extent of IBa decay at different drug concentrations during depolarizing test pulses to 20 mV (26). As for the L-type construct Lc, (+)-cis-diltiazem blockade of mutants I1487A and V1504A occurred with bimolecular association kinetics. The drug association rate (kon) for all three constructs was proportional to the drug concentration, and the off rate (koff) was independent of the applied drug concentration (Fig. 5). We observed no differences between the drug association kinetics of Lc and mutant I1487A. However, V1504A displayed significantly slowed association kinetics compared with Lc with kon(Lc) = (5.85 ± 0.24) × 103 M-1 s-1 versus kon(V1504A) = (4.93 ± 0.24) × 103 M-1 s-1 (p < 0.005). No significant changes in koff were observed for the Lc mutants.


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Fig. 5.   IBa inhibition of Lc and derived mutants I1487L, I1487A, and V1504A at 20 mV. The rate constants (kon and koff) of an open channel block by (+)-cis-diltiazem were estimated from the current relaxation time course (tau , see single exponential fit on currents in the inset: 1, control; 2, in 30 µM (+)-cis-diltiazem; 3, in 100 µM (+)-cis-diltiazem; and 4, in 300 µM (+)-cis-diltiazem). The extent of block (f = Isteady-state/Ictrl) was estimated during the first test pulse after 3 min incubation of the oocyte in drug. koff was estimated as f/tau . Data points are the mean values with standard error of 5-7 experiments. Linear regression yielded kon (Lc)= (5.85 ± 0.24) × 103 M-1 s-1, kon (I1487A)= (6.1 ± 0.14) × 103 M-1 s-1 and kon (V1504A) = (4.93 ± 0.24) × 103 M-1 s-1 (R > 0.98). Mean koff did not depend on drug concentration and ranged between 0.16 and 0.27 s-1.


    DISCUSSION

The characteristics of stereospecific and use-dependent L-type Ca2+ channel block by diltiazem can be transferred to the lowly sensitive alpha 1A subunit of class A Ca2+ channels by three L-type specific amino acids that have previously been identified as high affinity determinants of the PAA receptor site (11, 12). These experiments suggested that the BTZ and PAA receptors have overlapping but not identical determinants in the Ca2+ channel segment IVS6. Cai et al. (13) reported an approximately 10-fold increase in the half-maximal inhibitory concentration of Ca2+ currents by diltiazem if the PAA receptor determinants in segments IVS6 of an L-type channel (alpha 1C-a) were replaced by the corresponding alpha 1A amino acids. Here we confirm a key role of segment IVS6 for Ca2+ channel interaction with (+)-cis-diltiazem and report additional BTZ-specific receptor determinants in this pore lining segment (Fig. 2).

Structural Determinants of Resting State-dependent Action of (+)-cis-Diltiazem-- Our data show that an amino acid residue (Ile1487), which is located close to the extracellular mouth of the L-type channel pore, as well as an inactivation determinant (Val1504; alpha 1C-a numbering) at the inner channel mouth participate in the regulation of Ca2+ channel block by BTZs. As shown in Fig. 4, an alanine or leucine substitution of Ile1487 in Lc (compare also AL1 and L1383I, Fig. 3) substantially accelerated channel unblock at rest (Figs. 3 and 4) without altering the association or dissociation rate of (+)-cis-diltiazem for open channels (Fig. 5). The reduction in use-dependent block in the Lc mutants can therefore be explained by an enhanced recovery of channels between the individual pulses of a train rather than by a reduced drug affinity of the channels in the open state.

Enhanced recovery could be caused by a reduced bulkiness of a single amino acid resulting in facilitated drug dissociation or alternatively by a direct interaction of the drug molecule with the amino acid side chain in the resting state. If the bulkiness of a single isoleucine in position 1487 would restrict dissociation of (+)-cis-diltiazem from its receptor, removal of the side chain by the substituting isoleucine with the substantially smaller alanine is expected to facilitate channel unblock to a greater extent than exchanging isoleucine for the equally bulky and hydrophobic leucine. However, both Leu and Ala reduced use-dependent Ca2+ channel block to a comparable extent (Fig. 4). Furthermore, channel unblock kinetics of I1487L and I1487A at rest were indistinguishable and occurred at a significantly faster rate than unblock of Lc (Fig. 4, C and D). It is therefore unlikely that the enhanced unblock at rest is determined by the bulkiness of a single amino acid in position 1487. Instead, a single methyl group appears to form part of the BTZ-binding pocket of resting closed channels.

It is hard to distinguish whether this methyl group exclusively contributes to binding of diltiazem to closed resting channels or additionally forms part of a complex guarding structure. In the L-type environment of Lc, amino acid substitutions I1487L and I1487A did not significantly decrease resting state-dependent block (Fig. 4B). Therefore, we favor a "guarding hypothesis" where I1487 simultaneously forms part of a guarding structure. It is tempting to speculate that this guarding structure determines not only channel unblock (Figs. 2, 3, and 4C) but also access of (+)-cis-diltiazem to its binding determinants (Fig. 6).


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Fig. 6.   A guarded and modulated BTZ receptor. Putative pore orientated amino acids in the central part of transmembrane segment IVS6 contribute high affinity determinants of the BTZ and PAA receptor sites (Tyr1490, Ala1494, and Ile1497; alpha 1C-a numbering; see Refs. 11-13). Amino acid substitutions at position 1487 affect channel unblock at rest (Figs. 3B and 4, C and D) and consequently use-dependent block during a train (Figs. 3A and 4B). Our data suggest that isoleucine in position 1487 of alpha 1C-a (close to the extracellular channel mouth) forms part of the BTZ receptor site and additionally determines dissociation and access of (+)-cis-diltiazem to its receptor site in the closed resting state. Other inactivation determinants close to the inner channel mouth in segment IIIS6 (Phe1191 and Val1192; alpha 1C-a nomenclature) also affect use-dependent Ca2+ channel block by BTZ (15) and PAA (24). Val1504 at the inner mouth of segment IVS6 is a strong L-type channel inactivation determinant (Fig. 4A) simultaneously affecting channel block by (+)-cis-diltiazem (Figs. 4B and 5). The role of Val1504 in channel block can be explained by an allosteric modulation of the drug binding reaction. Additional trapping of the blocker in an inactivated channel state or a direct contribution of binding energy by Val1504 cannot be excluded.

Our data suggest that the BTZ receptor is located closer to the extracellular mouth of the channel pore than the PAA receptor (11, 12), which is in line with previous findings demonstrating an extracellular access of quaternary BTZ SQ32,428 (27) compared with the intracellular access of PAA (25, 28, 29).

Modulation of the BTZ Receptor by a Channel Inactivation Determinant-- A cluster of homologous inactivation determinants located close to the inner mouth of the channel pore affecting Ca2+ channel block by PAA was recently identified in segment IIIS6 (24). Kraus et al. (14) reported that alanine substitutions of the same residues in segment IIIS6 of a BTZ-sensitive class A/L-type channel chimera decreased both current inactivation and use-dependent block by (+)-cis-diltiazem.

Here we report an analogous role of a residue at the inner mouth of the pore in segment IVS6 in a recombinant L-type channel. As shown in Fig. 4A, substituting a single valine in segment IVS6 by alanine (V1504A) dramatically slowed Ca2+ channel inactivation kinetics and reduced use-dependent block (Fig. 4, A and B). It is interesting to note that the inactivation determinant valine in position 1504 of Lc (Val1504) is conserved in all known Ca2+ channel classes with the exception of the alpha 1S subunit cloned from carp skeletal muscle (Fig. 1). These findings provide further evidence for a key role of S6 segments in inactivation gating of Ca2+ channels and confirm a crucial role of homologous residues that are located at the inner channel mouth (see Ref. 30 for review).

The analysis of (+)-cis-diltiazem interaction with the inactivation-deficient mutant V1504A at a depolarized potential revealed a reduced drug association rate (kon) compared with mutants I1487A and Lc (Figs. 4 and 5). The reduced use-dependent block of V1504A (Fig. 4B) is therefore caused by a reduced drug binding reaction at 20 mV and not by an accelerated channel unblock at rest as observed for I1487L and I1487A (Figs. 4D and 5). Once the drug gained access to its receptor determinants in the open state, inactivating channels are in a more favorable conformation for interaction with (+)-cis-diltiazem than the "noninactivating" mutant V1504A (Figs. 4A and 5).

A guarded and modulated receptor hypothesis illustrating the impact of Ile1478 at the outer channel mouth and Val1504 at the inner channel mouth in use-dependent Ca2+ channel block by (+)-cis-diltiazem is schematically illustrated in Fig. 6. Ile1487 and Val1504 are separated by five turns of the alpha -helical IVS6 segments (approx  27 Å). Compared with the short distance of the putative pharmacophores of diltiazem (between 6 and 9 Å; see Ref. 31) the extended structure of the BTZ receptor suggests that some of the inactivation determinants at the inner channel mouth of IIIS6 (15) and IVS6 (Fig. 4) affect Ca2+ channel block not by contributing binding energy but via allosteric modulation of the drug binding process. However, neither an additional drug trapping of (+)-cis-diltiazem in an inactivated channel state (24) nor a direct contribution of binding energy by Val1504 can be excluded (Fig. 6).

In summary, we describe for the first time determinants of the BTZ receptor site that selectively affect BTZ interaction with Ca2+ channels in resting and open or inactivated states. We have demonstrated that a mutation at the inner channel mouth reducing channel inactivation (V1504A) simultaneously reduces the affinity of the BTZ binding site at depolarized potentials. Mutations at the external mouth of the pore (I1487L and I1487A) modulate drug dissociation without significantly affecting the drug affinity of open channels. Further mutational analysis of drug receptor sites will require a differentiation between drug access pathways, drug-binding sites, and conformational changes during membrane depolarization (e.g. inactivation).

    ACKNOWLEDGEMENTS

We thank Drs. Y. Mori and K. Imoto for the gift of the alpha 1A cDNA, Dr. A. Schwartz for providing the alpha 1C-a and alpha 2/delta cDNA, and B. Kurka and D. Kandler for expert technical assistance. (+)-cis-Diltiazem was kindly provided by Dr. Satzinger (Goedecke AG, Germany).

    FOOTNOTES

* This work was supported Fonds zur Förderung der Wissenschaftlichen Forschung Grants S6603-MED (to S. H.), P12649-MED (to S. H.), and P12689-MOB (to H. G.), a grant of the Else Kröner Fresenius Stiftung (to S. H.), and funds from the Hans und Blanca Moser Stiftung (to S. A.).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 These authors contributed equally to this work.

§ To whom correspondence should be addressed. Tel.: 43-512-507-3154; Fax: 43-512-588627; E-mail: Steffen.Hering{at}uibk.ac.at.

    ABBREVIATIONS

The abbreviations used are: PAA, phenylalkylamine; BTZ, benzothiazepine; IBa, barium inward current through voltage-gated Ca2+ channels.

    REFERENCES
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Abstract
Introduction
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