David R. Ferry

Assay for calcium channels

Publisher Summary This chapter focuses on biochemical assays for Ca 2+ -selective channels in electrically excitable membranes, which are blocked in electrophysiological and pharmacological experiments by verapamil, 1,4-dihydro-pyridines, diltiazem (and various other drugs), as well as inorganic di- or trivalent cations. The strategy employed is to use radiolabeled 1,4-dihydropyridine derivatives, which block calcium channels with ED 50 values in the nanomolar range. The affinity for their drug receptor site within the calcium channel can be very high. Their interaction with other membrane components, including sodium channels or neurotransmitter receptors, is negligible, which makes the choice of tissue preparation and purity of the respective membrane fraction less critical. A spectrum of compounds exists that regulate calcium channel function from blockade to opening. The chapter also discusses the tissue specificity of channel labeling, the complex interactions of divalent cations with the nimodipine-labeled calcium channels, and the allosteric regulation of nimodipine binding by the optically pure enantiomers of phenylalkylamine and benzothiazepine calcium channel blockers.

Evidence for multiple receptor sites within the putative calcium channel

Summary[3H]-Nimodipine, a potent calcium channel blocker, binds to an apparently homogeneous population of receptors in guinea-pig brain membranes (KD=0.62 nM, Hill coefficient}1.0). Diltiazem (10−5 M) lowers the KD for [3H]-nimodipine by a factor of 3 without changing the maximum number of binding sites. Diltiazem decreased the dissociation rate constant of the nimodipine-receptor complex from 0.18 min−1 to 0.049 min−1 and altered the pharmacological profile as revealed by displacement studies with (−) and (+) verapamil, (−) and (+) prenylamine and 1,4 dihydropyridines. In conclusion [3H]-nimodipine binding can be utilized as a tool to evaluate complex molecular interactions between calcium channel blockers.

Resolving the structure of the Ca2+ channel by photoaffinity labelling

Abstract Structural data relating to the L-type Ca 2+ channel have not always been consistent between — or even within — different laboratories. In drawing together these data , Hartmut Glossmann and colleagues focus particularly on the arylazide 1,4-dihydropyridine, azidopine, which specifically photoincorporates into Ca 2+ channels and which has begun to clarify the confusion that abounds. They also discuss problems associated with the low affinity binding sites for 1,4-dihydropyridines which are often abundant compared to channel-linked . receptors.

Identification of Voltage Operated Calcium Channels by Binding Studies: Differentiation of Subclasses of Calcium Antagonist Drugs with 3H-Nimodipine Radioligand Binding

3H-Nimodipine (3H-NIM) is a high affinity radioligand suitable to study Ca2+ -channels in a variety of tissues. The binding is saturable, reversible, and stereospecific in purified bovine heart and partially purified guinea-pig brain membranes. In the latter a Bmax of 600fmol/mg protein, dissociation constants (KD) of 0.4-0.8nM and a Hill slope of 1.0 are found. At 37 degrees C the optimal pH in 50mM TRIS-HCl buffer is 7.1-7.4. The calcium channel is a metalloprotein, and the divalent cation which is essential for the binding of 3H-NIM can be removed by EDTA (EC50 20 microM); the nimodipine binding site of the channel may then be reconstituted by divalent cations with Mn2+ greater than Ca2+ greater than Mg2+ greater than Sr2+. Ca2+ -antagonist drugs can be divided into three main classes based on their interaction with the 3H-NIM binding site: Class I has one site law of mass action-displacement isotherms with 3H-NIM, Class II exhibits complex biphasic inhibition profiles and Class III drugs increase the affinity of 1,4 dihydropyridines for the Ca2+ -channel. Diltiazem is a Class III Ca2+ -antagonist. Our in vitro studies lead us to conclude that the Ca2+ -channel contains multiple regulatory sites at which drugs can act.

Differential labelling of putative skeletal muscle calcium channels by [3H]-nifedipine, [3H]-nitrendipine, [3H]-nimodipine and [3H]-PN 200 110

SummaryFour 1,4-dihydropyridine calcium channel blockers [3H]-nifedipine, [3H]-nitrendipine, [3H]-nimodipine and [3H]-PN 200 110 (Isopropyl 4-(2,1,3,benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-methoxy-carbonylpyridine-3-carboxylate) were employed to label putative calcium channels in guinea-pig hind limb skeletal muscle membranes.The four radioligands differed with respect to the number of sites (Bmax) which were labelled. The following rank order of Bmax values was found: PN 200 110 > nimodipine = nitrendipine > nifedipine. d-cis-Diltiazem caused an increase in the density of high-affinity binding sites for all four calcium channel blockers. The relative stimulation was smallest for PN 200 110.It is suggested that 1,4-dihydropyridines exhibit different efficacies for induction of a channel state with high affinity for these drugs.

[125I] N6-p-hydroxyphenylisopropyladenosine, a new ligand for Ri adenosine receptors

SummaryN6-p-Hydroxyphenylisopropyladenosine (HPIA) has been labelled with carrier-free Na[125I] to very high specific activity (2,175 Ci/mmol) and used as an agonist ligand to characterize Ri adenosine receptors in rat cerebral cortex membranes. The binding is saturable, reversible, stereospecific and dependent on protein concentration. The specific binding at 37°C was of high affinity with an equilibrium dissociation constant KD of 0.48 nmol/l and was saturable with 0.23 pmol of [125I]HPIA per mg of protein. The rate constant of association, k1, was 3.25×108 l mol−1 min−1 and that of dissociation, k2, 0.0110 min−1 yielding a t1/2 of 63 min. In competition experiments the (−)isomer of N6-phenylisopropyladenosine (PIA) was 16-fold more potent than the (+)isomer in competing for the binding sites. Specific binding was most effectively displaced by N6-cyclohexyladenosine (CHA, ki=0.26 nmol/l), (−)PIA (ki=0.33 nmol/l) and HPIA (ki=0.52 nmol/l), whereas 5′-N-ethylcarboxamidoadenosine (NECA, ki-1.42 nmol/l) was less effective. The methylxanthines 3-isobutyl-1-methylxanthine (IBMX), theophylline and caffeine which have been classified as adenosine antagonists had ki values between 5–34 μmol/l. Binding of [125I]HPIA was regulated by guanine nucleotides and divalent cations. The results indicate that [125I]HPIA labels Ri adenosine receptors in rat brain membranes.