Abstract
omega-Conotoxin-sensitive N-type calcium channels control neurotransmitter release at the nerve terminal and interact with proteins implicated in secretion. Solubilized omega-conotoxin receptors from rat brain synaptic membrane were immunoprecipitated by antibodies against calcium channel alpha 1 subunits, syntaxin, and a 105-kDa plasma membrane protein. A multimeric complex, composed of calcium channel subunits, and synaptic proteins that showed varying degrees of association, was purified by a procedure involving anti-syntaxin immunoaffinity chromatography. A 250-kDa N-type alpha 1 subunit, containing cAMP-dependent phosphorylation site(s), was identified by photoaffinity labeling with 125I-azidonitrobenzoyl omega-conotoxin and immunoblotting with sequence-directed antibodies. An immunologically related 210-kDa form of the alpha 1 subunit was detected that displayed different pharmacological and regulatory properties. Protein bands of 140, 70, 58, and 35 kDa comigrated with purified alpha 1 subunits upon sucrose gradient centrifugation, whereas the 105-kDa protein was removed. The 58- and 35-kDa bands contained, respectively, the synaptic vesicle protein synaptotagmin and syntaxin, a plasma membrane protein that binds synaptic vesicle proteins. Purified omega-contoxin receptors were quantitatively immunoprecipitated by anti-syntaxin antibodies. These proteins may constitute an isolated exocytotic complex in which the N-type calcium channel tightly interacts with a synaptic vesicle docking site.
Highlights
From the Unstitut National de la Santd et de la Recherche Mbdicale U374, Znstitut Jean Roche, Facultd de Mea2cine
Solubi
Protein bandofs140,70,58, and 35 kDa comi- al.,1992), and form a ternary complex containing the plasma grated with purifieda1 subunits upon sucrose gradient membrane protein syntaxin (Bennetett al., 1992;Yoshida et al, centrifugation,whereasthe 106-kDa proteinwasre- 1992a; and Morita et al.,1992).Syntaxin interactsdirectly with moved.The 58- and35-kDabandscontained,respecsynaptotagmin (Bennettet al., 1992), and it can thusprovide a tively, the synaptic vesicle protein synaptotagmin and receptor for synaptic vesicles at the plasma membrane
Summary
(Received for publication, September 10, 1993, and in revised form, November 8, 1993). Purified o-conotoxin receptors pwepetridees in which synaptotagmin is peripherally associated quantitatively immunoprecipitated by anti-syntaxin awni-th a tightly interactingsyntaxiniN-type calcium channel core tibodies These proteins may constituatne isolated exo- structure. The supernatant was batch incubated for 5 min with 60 ml of chelating Sepharose Fast Flow loaded with cobalt ions (CoC12)and equilibrated with 2 volumes of 0.3 M NaCl in buffer B (10 m Hepes, 0.4%CHAPS, 0.02%phosphatidylcholine adjusted to pH 7.4 with NaOH). The diluted heparin-Ultrogel fractions were loaded onto10H5-Sepharosea t 0.2 dmin.The column was washed with 40 volumes of 1 M NaCl in buffer C, followed by volumes H,O, and eluted with 0.1 M triethylamine, 0.2 M NaCl in buffer C adjusted to pH 10.5 with HCl. Eluted proteins were eitherdenatured or immediately adjusted to pH 7.4 by addition of 0.5 M HepedNaOH. 150plof 50 m Hepes, 10m MgCl2,1n w EDTA, pH 7.4containing 10 m catalytic subunit of cAMP-dependentprotein kinase and 10 pCi of [32P]ATP.After 10minat 32 "C the reaction was stoppedby addition of SDS
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