Diazipine, a novel photoaffinity probe for dihydropyridine receptors of calcium channels

Abstract

INTRODUCTLON 2, MATERIALS AND METHODSVoltage regulated L-type calcium channels are widely distributed in many excitable cells of tissues such as skeletal, cardiac, and smooth muscle or brain. These channels have high affinity binding sites for a variety of drugs, e.g. 1,4-dihydropyridines, phenylalkylamines, bcnzothiazepines, and diphenylbutylpiperidincs ctc, (see [I] for review). The 1,4-dihydropyridines (DHP) are among the most useful ligands to identify the Ltype calcium channels even in broken cell preparations, DHP binds to the a-1 subunit of the L-type channels [2]. Since the primary structures of cr-1 subunits from various tissues (skeletal, cardiac, and smooth muscle) are known [3-51, it is a nmjor goal to identify the DHP binding site(s) within the primary amino acid sequences, Photoaffinity labeling plays an essential role to achieve this goal. Previously we have reported several reagents [&?'I and toxin derivatives for specifically photolabeling the sodium channels [G-10]. Recently we have synthesized diazipine (Fig. l), a novel photoreactive derivative of DHP possessing a phenyldiazirine as a carbene precursor [ll]. In this paper we describe the properties of diazipine, in terms of binding and photolabeling of the a-1 subunit in purified preparation of caldium channels from rabbit skeletal muscles. By comparison with azidopine, the currently used photoreactive DHP [Ia] characteristics of diazipine in binding affinity, photoincorporation efficiency, and photoproduct stability will also be described. Bitrdirtg experinren tsInto the WGA-purified calcium channels (4~41: of protein) in 50 mM TrisXl (pH 7,4) containing 2 mM CaC12, IOOrM cisdiltiazcm, and 0.1% digitonin 'binding buffer', ("Hlcliazipine was added in various concentrations (l-80 nM) and final volumes were 5004, The mixture was incubated at 25% for I h in the dark, Two 100 cl aliquots of the incubates were loaded on a Scphadex G-50 (fine) column (2 ml) which was pre-equilibrated with the binding buffcr and gel-filtrated by ccntrifugation as described [l3]. Radioactivity of the filtrate was measured by scintillation counting, Binding in the presence of IO PM (&)PN200-1 IO was also carried out to measure non-specific binding, Duplicate runs were performed for each data point.For

Synthesis of nitrodiazirinyl derivatives of neurotoxin II fromNaja naja oxiana and their interaction with theTorpedo californica nicotinic acetylcholine receptor

“…The carbenes are shorter lived than the nitrenes formed from arylazides, which should result in a smaller number of labeled receptor fragments. Diazirine-derivativized neurotoxins were successfully used in studies with ion channels (Hatanaka et al, 1992;Nakayama et al, 1991;Taki, et al, 1991). In this paper we describe the synthesis of NT-II derivatives containing nitrodiazirine labels in different positions of its amino acid sequence and their interaction with the AChR.…”

Identification of 1,4-dihydropyridine binding regions within the alpha 1 subunit of skeletal muscle Ca2+ channels by photoaffinity labeling with diazipine.

“…Both ligands possess similar reversible binding properties for skeletal muscle Ca2`-channel-associated DHP receptors (12). In contrast to [3H]azidopine, the covalent bond formed between [3H]diazipine and the receptor site is chemically stable against acid treatment and reducing agents (12,13). Fig.…”

“…Knowledge of the regions of the al subunit involved in the formation of the DHP receptor site would help to elucidate the molecular basis of channel modulation by DHPs and allosteric interaction between the DHP and phenylalkylamine binding domains. In this report, we define the location of the DHP binding domain within the al subunit of skeletal muscle Ca2" channels by photoaffinity labeling with the DHP-receptor-selective ligand trifluoroethyl diazirine [3H]diazipine (12,13) and mapping of labeled proteolytic fragments using sequence-directed antibodies.…”

Dihydropyridine receptor of L-type Ca2+ channels: identification of binding domains for [3H](+)-PN200-110 and [3H]azidopine within the alpha 1 subunit.

“…Application of similar methods to identification of the regions of the al subunit photolabeled by the DHPs diazipine, azidopine, and PN200-110 is reported here and in a preceding paper (7). The three ligands possess almost identical binding properties to membrane-bound and purified L-type Ca2+ channels (8)(9)(10)(11)(12) but are structurally different. Diazipine and azidopine contain photoreactive diazirine and phenylazide groups, respectively, which are located on a side chain up to 14.3 A (11) from the DHP ring, the proposed binding center.…”