Cloning and sequence analysis of cDNA for the Electrophorus electricus electroplax sodium channel indicate that this protein, consisting of 1,820 amino acid residues, exhibits four repeated homology units, which are presumably oriented in a pseudosymmetric fashion across the membrane. Each homology unit contains a unique segment with clustered positively charged residues, which may be involved in the gating structure, possibly in conjunction with negatively charged residues clustered elsewhere.

A series of simple methods for modifying diazirines bearing an aromatic ring has been accomplished. This first versatile approach involving direct substitution on the aromatic ring of diazirines has been achieved by means of the aromatic thallation of (alkoxyphenyl)diazirines. Introduction of the thallium moiety was successfully followed by nitration, iodination, or palladium-catalyzed carbonylation to give a family of substituted aryldiazirines useful for photolabeling. For instance, diazirines labeled with a nitro group can be detected by spectrophotometric methods, and those labeled with an iodo group can be useful in tracer experiments. The (methoxyphenyl)diazirines were also found to be stable under certain demethylation conditions, thus providing a potential source of diazirines with modifiable phenol hydroxyl groups. By means of this approach, a spacer arm to link diazirines with ligands was readily introduced. Radioactive diazirines labeled with carbon-14 or tritium were also prepared using this method. All the new diazirines were derived from a pair of simple (methoxyphenyl)diazirines. The ease of derivatization of the (alkoxyphenyl) diazirines described here may offer a practical approach to simplify the time-consuming methods currently used for diazirine synthesis.

To identify regions that are involved in the formation of the dihydropyridine receptor site of skeletal muscle L-type Ca2+ channels, the al subunit of the channel complex was specifically labeled with the 1,4-dihydropyridinereceptor-selective photoaffinity probe [3Hjdiazipine. Photoaffinity-labeled regions were identified by probing labeled proteolytic fragments with several anti-peptide antibodies recognizing different segments of the al sequence. Forty to 50% of the al-associated [3Hldiazipine label was contained in the tryptic fragment between Arg-988 and Ala-1023 derived from the loop between segments S5 and S6 in domain HI. This region corresponds to a portion of the channel that is believed to contribute to formation of the transmembrane pore. Twenty to 30% of the labeling occurred in a V8 protease fragment between Glu-1349 and Trp-1391. This fragment contains transmembrane segment S6 of domain IV and has previously been shown to form part of the drug receptor for phenylalkylamine Ca2+ antagonists. Our data suggest that the dihydropyridine receptor is formed by close apposition of two discontinuous regions of the al subunit sequence in domains Im and IV. In light of previous work localizing this receptor site to the extracellular surface of the lipid bilayer, it is proposed that amino acid residues at the extracellular surface in the loop connecting segments mSs and IIIS6 and at the extracellular end of segment IVS6 contribute to formation of the dihydropyridine receptor site. These drugs specifically bind with high affinity to distinct allosterically coupled receptor sites on the al subunit of the calcium channel complex as revealed by photoaffinity labeling (4-7). Neutron-diffraction and kinetic studies (8) suggest that DHPs approach their receptor site by lateral diffusion after partition into the lipid bilayer of the membrane. This binding domain is thought to be localized close to the outer surface of the al subunit as DHPs in a membraneimpermeable charged form only have access to their receptor site if applied from the extracellular side (9, 10). This is in contrast to phenylalkylamine Ca2+ antagonists, such as verapamil, that interact with an intracellular receptor site formed in part by the intracellular end of transmembrane helix IVS6 and/or the adjacent intracellular amino acid residues (11). 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.

Recently, we synthesized a new fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM) which is nonfluorescent by itself but will react readily with-SH groups to form highly fluorescent addition products. By the use of this reagent, we studied the localization and concentration of-SH groups and S-S linkages in the human epidermis. The distribution of-SH groups in living layers was abundant in cytoplasm but not in nuclei. The fluorescence was concentrated on the cell membrane or intercellular spaces (MIC parts) and was increased at the spino-granular junction. In the horny layer, the fluorescence of the MIC parts appeared brilliantly in the lower layers and decreased gradually. On the other hand, the fluorescence of cytoplasm in keratinized cells in the stratum corneum was faint. The localization of S-S linkages was not a characteristic of the living layers, but appeared abruptly at the junction of living and horny layers. The fluorescence was localized to the MIC parts and disappeared

Photochemistry in recent years has extended the armamentarium of the organic chemist in coping with synthetic challenges.1 Novel chromophoric systems have been developed and designed, and reactivities of excited states have been defined and utilized. The technique of selective quenching has made possible bond formation and fragmentation leading to molecular structures which would be not readily accessible by recourse to conventional reactions proceeding through ground-state paths.1,2 Probably the most active area of organic photochemistry has been the study of systems which possess a carbonyl group.3 As a result, a