Jörg Mitterdorfer

Transfer of High Sensitivity for Benzothiazepines from L-type to Class A (BI) Calcium Channels

To investigate the molecular basis of the calcium channel block by diltiazem, we transferred amino acids of the highly sensitive and stereoselective L-type (α1S or α1C) to a weakly sensitive, nonstereoselective class A (α1A) calcium channel. Transfer of three amino acids of transmembrane segment IVS6 of L-type α1 into the α1A subunit (I1804Y, S1808A, and M1811I) was sufficient to support a use-dependent block by diltiazem and by the phenylalkylamine (−)-gallopamil after expression in Xenopus oocytes. An additional mutation F1805M increased the sensitivity for (−)-gallopamil but not for diltiazem. Our data suggest that the receptor domains for diltiazem and gallopamil have common but not identical molecular determinants in transmembrane segment IVS6. These mutations also identified single amino acid residues in segment IVS6 that are important for class A channel inactivation.

Conserved Ca2+-antagonist-binding properties and putative folding structure of a recombinant high-affinity dihydropyridine-binding domain

Sensitivity to 1,4-dihydropyridines (DHPs) can be transferred from L-type (alpha1C) to non-L-type (alpha1A) Ca(2+) channel alpha1 subunits by the mutation of nine pore-associated non-conserved amino acid residues, yielding mutant alpha1A(DHP). To determine whether the hallmarks of reversible DHP binding to L-type Ca(2+) channels (nanomolar dissociation constants, stereoselectivity and modulation by other chemical classes of Ca(2+) antagonist drugs) were maintained in alpha1A(DHP), we analysed the pharmacological properties of (+)-[(3)H]isradipine-labelled alpha1A(DHP) Ca(2+) channels after heterologous expression. Binding of (+)-isradipine (K(i) 7.4 nM) and the non-benzoxadiazole DHPs nifedipine (K(i) 86 nM), (+/-)-nitrendipine (K(i) 33 nM) and (+/-)-nimodipine (K(i) 67 nM) to alpha1A(DHP) occurred at low nanomolar K(i) values. DHP binding was highly stereoselective [25-fold higher affinity for (+)-isradipine]. As with native channels it was stimulated by (+)-cis-diltiazem, (+)-tetrandrine and mibefradil. This suggested that the three-dimensional architecture of the channel pore was maintained within the non-L-type alpha1A subunit. To predict the three-dimensional arrangement of the DHP-binding residues we exploited the X-ray structure of a recently crystallized bacterial K(+) channel (KcsA) as a template. Our model is based on the assumption that the Ca(2+) channel S5 and S6 segments closely resemble the KcsA transmembrane folding architecture. In the absence of three-dimensional structural data for the alpha1 subunit this is currently the most reasonable approach for modelling this drug-interaction domain. Our model predicts that the previously identified DHP-binding residues form a binding pocket large enough to co-ordinate a single DHP molecule. It also implies that the four homologous Ca(2+) channel repeats are arranged in a clockwise manner.

Two Amino Acid Residues in the IIIS5 Segment of L-Type Calcium Channels Differentially Contribute to 1,4-Dihydropyridine Sensitivity

The transmembrane segment IIIS5 of the L-type calcium channel α1 subunit participates in the formation of the 1,4-dihydropyridine (DHP) interaction domain (Grabner, M., Wang, Z., Hering, S., Striessnig, J., and Glossmann, H. (1996) Neuron 16, 207-218). We applied mutational analysis to identify amino acid residues within this segment that contribute to DHP sensitivity. DHP agonist and antagonist modulation of Ba2+ inward currents was assessed after coexpression of chimeric and mutant calcium channel α1 subunits with α2δ and β1a subunits in Xenopus oocytes. Whereas DHP antagonists required Thr-1066, DHP agonist modulation crucially depended on the additional presence of Gln-1070 (numbering according to α1C-a), which also further increased the sensitivity to DHP antagonists. Asp-955, which is found at the corresponding position in the calcium channel α1S subunit from carp skeletal muscle, displayed functional similarity to Gln-1070 with respect to DHP interaction. We conclude that these residues (Thr-1066 plus Gln-1070 or Asp-955), which are located in close vicinity on the same side of the putative α-helix of transmembrane segment IIIS5, form a crucial DHP binding motif.

Molecular Basis of Drug Interaction with L-Type Ca2+ Channels

Different types of voltage-gated Ca2+ channels exist in the plasma membrane of electrically excitable cells. By controlling depolarization-induced Ca2+ entry into cells they serve important physiological functions, such as excitation-contraction coupling, neurotransmitter and hormone secretion, and neuronal plasticity. Their function is fine-tuned by a variety of modulators, such as enzymes and G-proteins. Block of so-called L-type Ca2+ channels by drugs is exploited as a therapeutic principle to treat cardiovascular disorders, such as hypertension. More recently, block of so-called non-L-type Ca2+ channels was found to exert therapeutic effects in the treatment of severe pain and ischemic stroke. As the subunits of different Ca2+ channel types have been cloned, the modulatory sites for enzymes, G-proteins, and drugs can now be determined using molecular engineering and heterologous expression. Here we summarize recent work that has allowed us to determine the sites of action of L-type Ca2+ channel modulators. Together with previous biochemical, electrophysiological, and drug binding data these results provide exciting insight into the molecular pharmacology of this voltage-gated Ca2+ channel family.

Calcium channels: The β‐subunit increases the affinity of dihydropyridine and Ca2+ binding sites of the α1‐subunit

A Ca2+ channel α1‐subunit derived from rabbit heart was transiently expressed in COS‐7 cells. The dihydropyridine (+)‐isradipine had low affinity (K i = 34.3 nM) for the α1‐subunit in the absence of the β‐subunit due to rapid dissociation (k −1 = 0.11 min−1). Co‐expression of the β‐subunit resulted in a ⪢ 35‐fold increase in (+)‐isradipine binding affinity (K i = 0.9 nM) due to decreased dissociation (k −1 of 0.007 min−1). Higher DHP binding affinity was associated with an increase of the apparent affinity of Ca2+ ions for the channel. Our data suggest that the β‐subunit affects the coordination of Ca2+ ions with sites that are coupled to the dihydropyridine binding domain and by this mechanism increases the affinity for these ligands.

Nine L-type Amino Acid Residues Confer Full 1,4-Dihydropyridine Sensitivity to the Neuronal Calcium Channel α1ASubunit ROLE OF L-TYPE MET1188

Pharmacological modulation by 1,4-dihydropyridines is a central feature of L-type calcium channels. Recently, eight L-type amino acid residues in transmembrane segments IIIS5, IIIS6, and IVS6 of the calcium channel α1subunit were identified to substantially contribute to 1,4-dihydropyridine sensitivity. To determine whether these eight L-type residues (Thr1066, Gln1070, Ile1180, Ile1183, Tyr1490, Met1491, Ile1497, and Ile1498; α1C-a numbering) are sufficient to form a high affinity 1,4-dihydropyridine binding site in a non-L-type calcium channel, we transferred them to the 1,4-dihydropyridine-insensitive α1A subunit using site-directed mutagenesis. 1,4-Dihydropyridine agonist and antagonist modulation of barium inward currents mediated by the mutant α1A subunits, coexpressed with α2δ and β1a subunits inXenopus laevis oocytes, was investigated with the two-microelectrode voltage clamp technique. The resulting mutant α1A-DHPi displayed low sensitivity for 1,4-dihydropyridines. Analysis of the 1,4-dihydropyridine binding region of an ancestral L-type α1 subunit previously cloned from Musca domestica body wall muscle led to the identification of Met1188 (α1C-a numbering) as an additional critical constituent of the L-type 1,4-dihydropyridine binding domain. The introduction of this residue into α1A-DHPi restored full sensitivity for 1,4-dihydropyridines. It also transferred functional properties considered hallmarks of 1,4-dihydropyridine agonist and antagonist effects (i.e. stereoselectivity, voltage dependence of drug modulation, and agonist-induced shift in the voltage-dependence of activation). Our gain-of-function mutants provide an excellent model for future studies of the structure-activity relationship of 1,4-dihydropyridines to obtain critical structural information for the development of drugs for neuronal, non-L-type calcium channels.

Differential Effects of Ca2+ Channel β1a and β2a Subunits on Complex Formation with α1S and on Current Expression in tsA201 Cells

To study the interactions of the α1S subunit of the skeletal musclel-type Ca2+ channel with the skeletal β1a and the cardiac β2a, these subunits were expressed alone or in combination in tsA201 cells. Immunofluorescence- and green fluorescent protein-labeling showed that, when expressed alone, β1a was diffusely distributed throughout the cytoplasm, β2a was localized in the plasma membrane, and α1S was concentrated in a tubular/reticular membrane system, presumably the endoplasmic reticulum (ER). Upon coexpression with α1S, β1a became colocalized with α1S in the ER. Upon coexpression with β2a, α1S redistributed to the plasma membrane, where it aggregated in large clusters. Coexpression of α1S with β1a but not with β2aincreased the frequency at which cells expressed l-type currents. A point mutation (α1S-Y366S) or deletion (α1S-Δ351–380) in the β interaction domain of α1S blocked both translocation of β1a to the ER and β2a-induced translocation of the α1S mutants to the plasma membrane. However, the point mutation did not interfere with β1a-induced current stimulation. Thus, β1a and β2a are differentially distributed in tsA201 cells and upon coexpression with α1S, form α1S·β complexes in different cellular compartments. Complex formation but not current stimulation requires the intact β interaction domain in the I-II cytoplasmic loop of α1S.

Mechanism of action of dexniguldipine-HCl (B8509−035), a new potent modulator of multidrug resistance

It has previously been shown that dexniguldipine-HCl (B8509-035) is a potent chemosensitizer in multidrug resistant cells [Hofmann et al., J Cancer Res Clin Oncol 118: 361-366, 1992]. It is shown here that dexniguldipine-HCl causes a dose-dependent reduction of the labeling of the P-glycoprotein by azidopine, indicating a competition of dexniguldipine-HCl with the photoaffinity label for the multidrug resistance gene 1 (MDR-1) product. Exposure to dexniguldipine-HCl results in a dose-dependent accumulation of rhodamine 123 in MDR-1 overexpressing cells. In the presence of 1 microM dexniguldipine-HCl, rhodamine 123 accumulated in multidrug resistant cells to similar levels as in the sensitive parental cell lines. At this concentration, dexniguldipine-HCl enhances the cytotoxicities of Adriamycin and vincristine. The resistance modulating factors (RMF), i.e. IC50 drug/IC50 drug + modulator, were found to be proportional to the expression of MDR-1, ranging from 8 to 42 for Adriamycin and from 16 to 63 for vincristine. Transfection with the MDR-1 gene was found to be sufficient to sensitize cells to the modulation by dexniguldipine-HCl. The compound does not affect the expression of the MDR-1 gene. Dexniguldipine-HCl has no effect on a multidrug resistant phenotype caused by a mutation of topoisomerase II. It is concluded that dexniguldipine-HCl modulates multidrug resistance by direct interaction with the P-glycoprotein.