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

Abstract

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.

Suppression of voltage-gated L-type Ca 2+ currents by polyunsaturated fatty acids in adult and neonatal rat ventricular myocytes

“…The finding of an inhibition of L-type Ca 2ϩ currents in the present study is more consistent with the earlier report that n Ϫ 3 PUFAs modulate the actions of dihydropyridine agonists and antagonists in cardiomyocytes (33). The ability of the n Ϫ 3 PUFAs, shown in that study, to displace [ 3 H]nitrendipine from its binding site at the extracellular pole of the calcium channel (34), suggests that the effects of PUFAs on I Ca,L result from noncovalent binding or interaction with the protein of the calcium channel as found with the voltage-sensitive sodium channel.…”

Tissue-specific expression of calcium channels

“…The 'extracellular" loop between transmembrane helices 5 and 6 (SSl-SS2 region) is predicted to fold into the membrane, to form part of the pore of the channel (Guy and Conti 1990), and to take part in the control of ion selectivity (Heinemann et al 1992). Photoaffinity labeling of skeletal muscle a, subunit followed by limited proteolysis and immunoprecipitation indicates that the DHP-binding site is localized close to the SSl-SS2 region of repeat III (Striessnig et al 1991, Nakayama et al 1991 and to a sequence following the IVS6 segment (Regulla et al 1991), whereas the PAAbinding site has been located directly after the IVS6 segment (Striessnig et al 1990) (Figure 2). Binding studies with radiolabeled DHPs demonstrate that the stably expressed al subunits from skeletal and smooth muscle alone contain the allosterically coupled binding sites for the known CaCBs (Kim et al 1990, Bosse et al 1992).…”

Azidobutyryl clentiazem, a new photoactivatable diltiazem analog, labels benzothiazepine binding sites in the α1 subunit of the skeletal muscle calcium channel

“…Peptide CP-(1011~1026C), CP-(GC1381-1399) or CP-(1401-1415CG) were synthesized by the addition of a cysteine, glycylcysteine, or cysteinylglycine residue to the C-or N-terminus of CP-(1011~1026C), CP-(1381-1399), CP-(1401-1415) respectively. Peptide synthesis, coupling to porcine thyroglobuhn, and antibody generation have been described [12].…”

“…Immunoprecipitation was carried out essentially as described [12]. Briefly, either digested or non-digested ['H]ABC-labeled a, was incubated with antibody-coupled protein A-Sepharose 4B-CL gel (100 ~1) in RIA buffer (10 mM Tris-HCl, pH 7.2 containing 150 mM NaCl and 0.3% Triton X-100) at room temperature.…”

“…The phenylalkylamine binding site is thought to be located at the intracellular surface of the membrane in the S6 segment of domain IV [l 11. In contrast, the dihydropyridine binding site is located extracellularly, near the putative channel pore involving transmembrane helices S6 in domain III and IV [12,13]. ***Visiting Scientist from Tanabe Seiyaku Co., Ltd. Pharmacological Research Laboratory, Saitama 335, Japan.…”