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[3H]HOE166 defines a novel calcium antagonist drug receptor — distinct from the 1,4 dihydropyridine binding domain

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Summary

Benzothiazinones represent a novel class of drugs which block voltage-dependent L-type calcium channels in different tissues. [3H]HOE166 (R-(±)-3,4-dihydro-2-isopro-opyl-4-methyl-2-[2-[4-[4-[2-(3, 4, 5-trimethoxyphenyl)ethyl]piperazinyl]butoxy]phenyl]-2H -1, 4-benzothiazin-3-on-dihydrochloride; ≈ 57 Ci/mmol) a potent optically pure benzothiazinone was employed to characterize receptors associated with skeletal muscle transverse tubule calcium channels. [3H]HOE166 reversibly labels the membrane-bound calcium channels with high affinity (Kd = 0.36 ± 0.05 nM; Bmax = 18.2 ± 3.3 pmol/mg of membrane protein; means ± SD, n = 13), HOE166 (Ki = 0.76 nM) is 29-fold more potent than the respective (S)-enantiomer (Ki = 22.1 nM). Binding is inhibited by divalent and trivalent cations (Cd2+ and La3+ being most potent) and other calcium channel drugs (1,4 dihydropyridines, phenylalkylamines, benzothiazepines). High affinity [3H]HOE166 binding activity is maintained (Kd = 4.5–9.0 nM) after solubilization and purification (554–1350 pmoles/mg of protein) of the calcium channel complex from transverse-tubule membranes. The following data support our recent claim (Striessnig et al. 1985, 1988) that HOE166 labels a domain on L-type calcium channels which is distinct from that defined by 1,4 dihydropyridines, phenylalkylamines or benzothiazepines: (1) All 1,4 dihydropyridine-, phenylalkylamine-and benzothiazepine-receptor-selective drugs tested are only very weak inhibitors of [3H]HOE166 binding. (2) (+)-PN200-110 only partially inhibits [3H]HOE166 binding to the purified calcium. channel complex. (3) The decay of the [3H]HOE166-receptor complex is monoexponential but the dissociation rate constants depend on the ligand concentration; (+)-PN200-100 accelerates the dissociation in the presence of unlabelled HOE166. (4) Nanomolar concentrations of HOE166 and HOE167 completely inhibit (−)-[3H]desmethoxyverapamil binding to a Drosophila phenylalkylamine receptor (which lacks a 1,4 dihydropyridine binding domain). Taken together, these results are incompatible with the view that [3H]HOE166 binds competitively to the calcium channel linked 1,4 dihydropyridine drug receptors.

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Abbreviations

kd:

dissociation constant

Ki :

inhibition constant

k−1,k+1 :

dissociation, association rate constant

SDS:

sodium dodecyl sulfate

T-tubule:

transverse tubule

s20,w :

sedimentation coefficient

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Authors and Affiliations

  1. Institut für Biochemische Pharmakologie, Universität Innsbruck, Peter Mayrstrasse 1, A-6020, Innsbruck, Austria

    Alfred Grassegger, Jörg Striessnig, Markus Weiler, Hans-Günther Knaus & Hartmut Glossmann

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  1. Alfred Grassegger
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  2. Jörg Striessnig
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  3. Markus Weiler
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  4. Hans-Günther Knaus
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  5. Hartmut Glossmann
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Grassegger, A., Striessnig, J., Weiler, M. et al. [3H]HOE166 defines a novel calcium antagonist drug receptor — distinct from the 1,4 dihydropyridine binding domain. Naunyn-Schmiedeberg's Arch Pharmacol 340, 752–759 (1989). https://doi.org/10.1007/BF00169685

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