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Anti-peptide antibodies as probes of the structure, function and distribution of Ca2+ channels

Brickley, Kieran David; (1994) Anti-peptide antibodies as probes of the structure, function and distribution of Ca2+ channels. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Fifteen peptides were synthesized corresponding to sequences in the primary structure of the a1 subunits of both the class A and class D rat brain, and of the a1 and a2 subunits of the rabbit skeletal muscle L-type Ca2+ channel. Conjugates of these peptides to various carrier proteins were used to produce polyclonal antibodies in rabbits. Each peptide-specific antibody was affinity purified and used to probe the structure, function and distribution of these channels. Nine of the antibodies identified (four strongly) their intact denatured polypeptide in t-tubules. The rat brain class A- specific antibody reacted strongly with a denatured polypeptide in skeletal muscle t- tubules, while only antibody N, raised against a sequence in a2, bound to its denatured polypeptide in brain membranes. Eight antibodies bound (four strongly) to the L-type Ca2+ channel in situ, in rabbit, rat, mouse and human skeletal muscle. One antibody reacted strongly, and three weakly with the corresponding channel in situ. in cardiac muscle cryosections from rabbit, rat and pig. Nine of the antibodies recognized (five very clearly) the intact, native, L-type channel purified from rabbit skeletal muscle. None of these antibodies showed any inhibition of binding of nitrendipine to the L-type Ca2+ channel in t-tubule membranes. The reactivity of the antibodies with the intact denatured channel polypeptides was examined in Western blots with rabbit and rat skeletal muscle t-tubules and brain membranes. In situ, identification of antibody binding was carried out using fluorescence immunocytochemistry in unfixed cryosections of skeletal muscle from rabbit, rat, mouse and human tissue and of cardiac muscle from rabbit, rat and pig. Antibody interaction with the native channel structure was studied by ELISA with the L-type channel following its purification from rabbit skeletal muscle. Inhibition of binding of either nitrendipine or D888 to the channel by the antibodies was assayed using radiolabelled ligand and t-tubule membranes which had been incubated with either test or control antibody. These antibodies have been used to confirm the predicted high sequence homology between rabbit skeletal muscle L-type Ca2+ channel a subunits and those found in rabbit, rat, human and mouse skeletal muscle, and in rat and porcine cardiac muscle, whose sequences are unknown. The IS4 domain, and peptides located on predicted intracellular loops and in the C-terminal region appeared to be exposed in the native α1 subunit. However, binding of dihydropyridines or phenylalkylamines to α1 was not affected by antibodies bound to any of the exposed α1 domains. Some β-sheet structure was revealed for a synthetic peptide, corresponding to the C-terminal α1(1390-1437) domain, in aqueous buffer using FTIR spectroscopic studies, L-type Ca2+ α polypeptides were located primarily to the transverse-tubule system of both skeletal and cardiac muscle. The relative abundance of the channel α2 subunit in rat brain membranes, was found to be not more than 10-30-fold less than that found in rat skeletal muscle transverse tubule membranes. The observed exposure of the IS4 domain in native a1 supports the model of the pore structure of voltage-gated ion channels, having both S2 and S4 as pore forming sequences, with the channel lined by the S5-S6 loop. Part of the loop between domain II and III in skeletal muscle α1, which has a role in excitation- contraction coupling is possibly inaccessible to antibody in situ, but becomes exposed during channel purification. A polypeptide, possibly a neuronal-type channel subunit was identified in t-tubules by the rat brain class A-specific antibody.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Anti-peptide antibodies as probes of the structure, function and distribution of Ca2+ channels
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
URI: https://discovery.ucl.ac.uk/id/eprint/10098884
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